(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date
31 January 2002 (31.01.2002)
PCT
(10) International Publication Number
WO 02/08748 A2
(51) International Patent Classification 7 : G01N 33/00
(21) International Application Number: PCT/US0 1/2 1652
(22) International Filing Date:
(25) Filing Language:
(26) Publication Language:
9 July 2001 (09.07.2001)
English
English
(30) Priority Data:
60/217,219
60/217,221
60/217,666
60/217,671
09/804,457
09/804,458
09/804,480
09/804,580
10 July 2000
10 July 2000
10 July 2000
10 July 2000
12 March 2001
12 March 2001
12 March 2001
12 March 2001
(10.07.
(10.07
(10.07
(10.07
(12.03
(12.03
(12.03
(12.03
.2000) US
2000) US
2000) US
.2000) US
.2001) US
.2001) US
2001) US
.2001) US
P. [US/US]; 10912CorteLuzdelSol, San Diego, CA 92130
(US). GONZALEZ, Jesus, E., Ill [US/US]; 12442 Caimel
Cape, San Diego, CA 92130 (US).
(74) Agent: ARNO, Thomas, R.; Knobbe, Martens, Olson &
Bear, LLP, 16th Floor, 620 Newport Center Drive, Newport
Beach, CA 92660 (US).
(81) Designated States (national): AE, AG, AL, AM, AT, AT
(utility model), AU, AZ, BA, BB, BG, BR, BY, BZ, CA,
CH, CN, CO, CR, CU, CZ, CZ (utility model), DE, DE
(utility model), DK, DK (utility model), DM, DZ, EC, EE,
EE (utility model), ES, FL FI (utility model), GB, GD, GE,
GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ,
LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN,
MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
SK, SK (utility model), SL, TJ, TM, TR, TT, TZ, UA, UG,
US, UZ, VN, YU, ZA, ZW.
(71) Applicant (/or all designated States except US): AURORA (84) Designated States (regional): ARIPO patent (GH, GM,
BIOSCIENCES CORPORATION [US/US]; HOlOTor-
reyanaRoad, San Diego, CA 92121 (US).
(72) Inventors; and
(75) Inventors/Applicants (for US only): MAHER, Michael,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
[Continued on next page]
(54) Title: ION CHANNEL ASSAY METHODS
(57) Abstract: A method of characterizing the biological activity of a candidate compound may include exposing cells to the can-
didate compound, and then exposing the cells to a repetitive application of electric fields so as to set the transmembrane potential to
a level corresponding to a pre-selected voltage dependent state of a target ion channel.
WO 02/08748 A2 llllllllllllllllllllllllllllllllllllllllllllllllllllllllll
Published: For two-letter codes and other abbreviations, refer to the "Guid-
without international search report and to be republished ance Notes on Codes and Abbreviations" appearing at the begin-
upon receipt of that report ning of each regular issue of the PCT Gazette.
WO 02/08748
PCT/US01/21652
ION CHANNEL ASSAY METHODS
Cross Reference to Related Applications
This application claims priority to U.S. Provisional Application Serial Nos. 60/217,671,
5 entitled INSTRUMENTATION AND METHODS FOR ELECTRICAL STIMULATION, filed on
July 10, 2000, by Maher et al.; 60/217,666, entitled INSTRUMENTATION AND METHODS
FOR ELECTRICAL STIMULATION, filed on July 10, 2000, by Mendlein; 60/217,221, entitled
INSTRUMENTATION AND METHODS FOR ELECTRICAL STIMULATION, filed on July 10,
2000, by Maher et al.; 60/217,219, entitled INSTRUMENTATION AND METHODS FOR
10 ELECTRICAL STIMULATION, filed on July 10, 2000, by Maher et al.; and U.S. Application
Serial Nos. 09/804,457, entitled ION CHANNEL ASSAY METHODS, filed March 12, 2001, by
Maher et al.; 09/804,480, entitled ION CHANNEL ASSAY METHODS, and filed March 12, 2001,
by Maher et al.; 09/804,580, entitled HIGH THROUGHPUT METHOD AND SYSTEM FOR
SCREENING CANDIDATE COMPOUNDS FOR ACTIVITY AGAINST TARGET ION
15 CHANNELS, filed March 12, 2001, by Maher et al.; and 09/804,458, entitled MULTI-WELL
PLATE AND ELECTRODE ASSEMBLIES FOR ION CHANNEL ASSAYS, filed
March 12, 2001.
Background of the Invention
Field of the Invention
20 The present invention relates generally to instrumentation and methods for manipulating
membrane potentials of living cells via electrical stimulation.
Description of the Related Art
It has long been known that the interior of animal and plant cells is electrically negative
with respect to the exterior. The magnitude of this potential difference is generally between 5 and
25 90 mV, with most of the potential being developed across the cell membrane. The transmembrane
potential of a given cell is set by the balance of the activities of ion transporters which create and
maintain the electrochemical gradient, and the activities of ion channels, passive diffusion and
other factors, that allow ions to flow through the plasma membrane.
Ion channels participate in, and regulate, cellular processes as diverse as the generation
30 and timing of action potentials, energy production, synaptic transmission, secretion of hormones
and the contraction of muscles, etc. Many drugs exert their specific effects via modulation of ion
channels. Examples include antiepileptic compounds like phenytoin and lamotrigine, which block
voltage-dependent sodium channels in the brain, antihypertensive drugs like nifedipine and
diltiazem, which block voltage-dependent calcium channels in smooth muscle cells, and
35 stimulators of insulin release like glibenclamide and tolbutamide, which block ATP-regulated
potassium channels in the pancreas.
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WO 02/08748 PCT/US01/21652
Finding new drugs which have specific modulatory effects on ion channels requires methods
for measuring and manipulating the membrane potential of cells with the ion channels present in the
membrane. A number of methods exist today that can be used to measure cell transmembrane
potentials and to measure the activities of specific ion channels. Probably the best known approach is
5 the patch clamp, originally developed by Neher, Sakmann, and Steinback. (The Extracellular Patch
Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological
Membranes, Pfluegers Arch. 375; 219-278, 1978). Other methods include optical recording of
voltage-sensitive dyes (Cohen et al., Annual Reviews of Neuroscience 1: 171-82, 1978) and
extracellular recording of fast events using metal (Thomas et al., Exp. Cell Res. 74: 61-66, 1972) or
10 field effect transistors (FET) (Fromherz et al., Science 252: 1290-1293, 1991) electrodes.
The patch clamp technique allows measurement of ion flow through ion channel proteins
and the analysis of the effect of drugs on ion channels function. In brief, in the standard patch
clamp technique, a thin glass pipette is heated and pulled until it breaks, forming a very thin (< 1
jum in diameter) opening at the tip. The pipette is filled with salt solution approximating the
1 5 intracellular ionic composition of the cell. A metal electrode is inserted into the large end of the
pipette, and connected to associated electronics. The tip of the patch pipette is pressed against the
surface of the cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel
proteins in a tiny patch of membrane. The activity of these channels can be measured electrically
(single channel recording) or, alternatively, the patch can be ruptured allowing measurements of
20 the combined channel activity of the entire cell membrane (whole cell recording).
During both single channel recording and whole-cell recording, the activity of individual
channel subtypes can be further resolved by imposing a voltage clamp across the membrane.
Through the use of a feedback loop, the voltage clamp imposes a user-specified potential
difference across the membrane, allowing measurement of the voltage, ion, and time dependencies
25 of various ion channel currents. These methods allow resolution of discrete ion channel subtypes.
A major limitation of the patch clamp technique as a general method in pharmacological
screening is its low throughput. Typically, a single, highly trained operator can test fewer than ten
compounds per day using the patch clamp technique. Furthermore the technique is not easily
amenable to automation, and produces complex results that require extensive analysis by skilled
30 electrophysiologists. By comparison, the use of optical detection systems provides for
significantly greater throughput for screening applications (currently, up to 100,000 compounds
per day), while at the same time providing for highly sensitive analysis of transmembrane
potential. Methods for the optical sensing of membrane potential are typically based on
translocation, redistribution, orientation changes, or shifts in spectra of fluorescent, luminescent,
35 or absorption dyes in response to the cellular membrane potential (see generally Gonzalez, et al.,
Drug Discovery Today 4:43 1-439, 1999).
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WO 02/08748 PCT/US01/21652
A preferred optical method of analysis has been previously described (Gonzalez and Tsien,
Chemistry and Biology 4: 269-277, 1997; Gonzalez and Tsien, Biophysical Journal 69: 1272-1280,
1995; and U.S. Patent No 5,661, 035 issued August 26, 1997). This approach typically comprises
two reagents that undergo energy transfer to provide a ratiometric fluorescent readout that is
5 dependent upon the membrane potential. The ratiometric readout provides important advantages
for drug screening including improved sensitivity, reliability and reduction of many types of
experimental artifacts.
Compared to the use of a patch clamp, optical methods of analysis do not inherently
provide the ability to regulate, or clamp, the transmembrane potential of a cell. Clamping methods
10 are highly desirable because they provide for significantly enhanced, and more flexible methods of
ion channel measurement. A need thus exists for reliable and specific methods of regulating the
membrane potentials of living cells that are compatible with optical methods of analysis and are
readily amendable to high throughput analysis.
Summary of the Invention
15 In one embodiment, a method of assaying ion channel activity comprises exposing at least
one cell to a plurality of electric field pulses so as to create a controlled change in transmembrane
potential and so as to activate an ion channel of interest, and detecting ion channel activity by
detecting one or more changes in transmembrane potential without using a patch clamp. The
monitoring may comprise detecting fluorescence emission from an area of observation containing
20 the cells. In some advantageous embodiments, the electric fields are biphasic.
In another embodiment, the invention comprises a method of characterizing the biological
activity of a candidate compound. The method includes exposing one or more cells to said
compound, repetitively exposing said one or more cells to one or more electric fields so as to effect
a controlled change in transmembrane potential of said one or more cells, and monitoring, without
25 using a patch clamp, changes in the transmeln one embodiment, a method of assaying ion channel
activity comprises exposing at least one cell to a plurality of electric field pulses so as to create a
controlled change in transmembrane potential and so as to activate an ion channel of interest, and
detecting ion channel activity by detecting one or more changes in transmembrane potential
without using a patch clamp.
30 Advantageously, pulsed biphasic electric fields may be used that have a maximum
amplitude of less than approximately 90 V/cm, are applied at a rate of at least about 1 per second,
and which have total duration of at least about 1 millisecond.
In another embodiment, cells are used in an ion channel assay method that express both an
ion channel of interest and a counter ion channel.
35 Methods and systems of compound screening are provided. In one embodiment, such a
method comprises expressing the target ion channel in a population of host cells and placing a
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WO 02/08748 PCT/US01/21652
plurality of the host cells into each of a plurality of sample wells. A candidate drug compound is
added to at least one of the plurality of sample wells; and the transmembrane potential of the cells
is modulated with a repetitive application of electric fields so as to set the transmembrane potential
to a level corresponding to a pre-selected voltage dependent state of the target ion channel.
5 Apparatus for high throughput screening is also provided. In one specific embodiment, a
plurality of wells having a high transmittance portion through which cells present in the wells are
optically observable in an area of observation are each provided with two electrodes. A power
supply is connected to the electrodes; wherein the power supply and the electrodes are configured
to apply a series of electric fields to cells within the area of observation, the electric fields having a
10 spatial variation of less than about 25% of a mean field intensity within the area of observation, the
electric fields being effective to controllably alter the transmembrane potential of a portion of the
cells. In addition, an optical detector is configured to detect light emanating from the wells
through the high transmittance portion, and a data processing unit is provided to interpret the light
emanating from the wells through the high transmittance portion as ion channel activity resulting
15 from the transmembrane potential alterations.
In one embodiment of the invention, an assay plate and electrode assembly comprises at
least one sample well having electrodes placed therein. The electrodes are positioned with respect
to the bottom surface of the well to provide an electric field adjacent to the bottom surface that
varies by less than about 10% from a mean field intensity over at least about 20% of the surface
20 area of the bottom surface.
Additional electrode/plate combinations of the invention include a bottom panel for a
multi-well plate comprising at least one row of high transmittance regions with positions
corresponding to well locations having a first strip of conductive material extending along the row
and overlapping a first portion of the well locations, and having a second strip of conductive
25 material extending along the row and overlapping a second portion of the well locations.
In another embodiment, an assay apparatus comprises a sample well, a first pair of
electrodes positioned within the sample well, and at least one additional satellite electrode
positioned within the sample well.
Brief Description of the Drawings
30 FIG. 1 Shows one embodiment of a dipper electrode array.
FIG. 2 Shows a number of embodiments of multiwell plates comprising surface
electrodes.
FIG. 3 Shows a block diagram of one embodiment of the electrical stimulation system.
FIG. 4. Shows the simulated effects of repetitive external electrical fields on a cell
35 expressing a voltage dependent sodium channel. The upper panel indicates the applied electrical
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WO 02/08748 PCT/US01/21652
field, the middle panel indicates the simulated sodium current into the cell, and the lower panel
indicates the simulated average transmembrane potential.
FIG. 5 Shows a schematic representation of a square wave.
FIG. 6 Shows examples of various wave kernels.
5 FIG. 7 Shows calculated electric field profiles for various electrode assemblies in round,
6.2 mm diameter wells. Dashed circle is a 3 mm diameter view window. In white areas, the
electric field strength is less than 10% of the average electric field strength in the view window. In
gray areas, the electric field strength is within 1 0% of the average electric field strength in the view
window. In black areas, the electric field strength is greater than 1 0% of the average electric field
1 0 strength in the view window.
FIG. 8 Shows calculated electric field profiles for various electrode assemblies in round
and square wells 6.2 mm across. Dashed circle is a 3 mm diameter view window. In white areas,
the electric field strength is less than 1% of the average electric field strength in the view window.
In gray areas, the electric field strength is within 1% of the average electric field strength in the
15 view window. In black areas, the electric field strength is greater than 1% of the average electric
field strength in the view window.
FIG. 9 Shows various electrode and insulator designs for improving electric field
uniformity in round wells.
FIG. 10 Shows the effect of electrical stimulation protocols at varying pulse amplitudes
20 over the time course of electrical stimulation in wild-type CHO cells.
FIG. 11 Shows the relationship between the maximal cellular response and the applied
pulse amplitude during electrical stimulation for wild-type CHO cells. Data was from FIG. 10
taken after about 5 seconds.
FIG. 12 Shows the dose response curve for the effect of TTX in wild-type CHO cells.
25 Stimulation parameters were 33 V/cm, 50 Hz for 3 seconds with a biphasic square wave kernel (5
ms per phase). The solid line is a Hill function fit to the data with EC50 = 9 nM and a Hill
coefficient of 1 .47.
FIG. 13 Shows the relationship between pulse duration and frequency and the cellular
response wild-type CHO cells during electrical stimulation. The electric field strength was always
30 25 V/cm. The stimulus was a three- second burst of biphasic pulses of varying duration and
Af
frequency. Solid lines are fits to the form R = 1 H — .
f fo
FIG. 14 Shows time traces for CHO cells expressing the NaV2 sodium channel cells
electrically stimulated at various field strengths. Cells were stimulated in a 96-well plate, with a
20 Hz, 3 second-long train of biphasic, 5 ms/phase voltage pulses. The stimulation occurred during
35 the shaded portion of the graph. In this experiment, the cells were stained with 20 \xM CC2-DMPE
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WO 02/08748 PCT/US01/21652
and 63 nM DiSBACg(3). This dye combination has a 2 ms time constant and accurately tracks the
transmembrane potential. The rise and fall times of the response were fitted to exponential decay
functions and were found to be x r i se =200 ms and Tf a n =850 ms.
FIG. 15 Shows the relationship between the electric field strength and the cellular
5 response measured after 4 seconds (squares) and 10 seconds (circles) of electrical stimulation. The
line is a Boltzman fit to the data.
FIG. 16 Shows the effect of pulse duration and stimulation frequency on the cellular
response of CHO cells expressing the NaV2 sodium channel.
FIG. 17 Shows the knee time parameter Tq from the fits to the data in FIG. 16 plotted
1 0 versus the stimulus duration.
FIG. 18 Shows the temporal response of HEK-293 cells expressing the NaV3 sodium
channel during electrical stimulation.
FIG. 19 Shows dose response curves for tetrodotoxin (FIG. 19A) and tetracaine
(FIG. 19B) for HEK-293 expressing the NaV3 sodium channel. Electrical stimulation conditions
15 were: E=33 V/cm, 10 ms/phase biphasic stimulation, 15 Hz burst for 1.5 seconds.
FIG. 20 Shows a dose response curve for tetracaine for HEK-293 expressing the NaV4 ion
channel. For this experiment, electrical stimulation parameters were E=33 V/cm, 10 ms/phase
biphasic stimulation, 15 Hz burst for 1.5 seconds.
FIG. 21. Shows a full-plate view of electrical stimulation of wild-type HEK-293 cells.
20 Each individual panel represents the time trace of the normalized fluorescence ratio of a single well
in the 96-well plate. Each well in a vertical column was stimulated simultaneously with the same
field strength. Field strength increases from left to right. Rows 6-8 contained 10 mM TEA to
block the voltage-dependent potassium channels.
FIG. 22. Shows the cellular response as a function of the stimulus field for wild type
25 HEK. Error bars are standard deviations. Open symbols: no added blockers. Filled symbols: 10
mM TEA added to block potassium channels.
FIG. 23 Shows the time response traces for selected concentrations of the sodium channel
blockers tetrodoxin (TTX) (FIG. 23A) and tetracaine (FIG. 23B) in CHO cells expressing the
NaV2 sodium channel.
30 FIG. 24 Shows the dose response curves for TTX and tetracaine inhibition of the NaV2
sodium channel.
FIG. 25. Shows a 'Random' TTX spiking experiment. Each small box in this 1 1x8 array
, contains the ten-second time trace of a well at the corresponding position of a 96-well plate. The
twelfth column was a control well without cells used for background subtraction and is not shown.
35 Wells (1,1), (2,2), (3,3), etc. contained a blocking concentration of TTX.
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WO 02/08748 PCT/US01/21652
FIG. 26 Shows an analysis of the 'random 5 TTX spiking data shown in FIG. 25. The data
points are the ratiometric response in the time window from 1.8-2.4 seconds after the beginning of
the stimulus burst (i.e. at the peak of the response). The filled circles points were spiked with 1
jliM TTX; the open circles had no blocker added.
5 FIG. 27. Shows a full-plate view of electrically-stimulated HL5 cardiac muscle cells.
Each individual panel represents the time trace of the normalized fluorescence ratio of a single well
in the 96-well plate. Each well in a vertical column was stimulated simultaneously with the same
field strength. Field strength increases from left to right. Rows 5 and 6 contained 10 |iM TTX to
partially block the voltage-dependent sodium channels. Rows 7 and 8 contained 10 raM TEA to
10 partially block the voltage-dependent potassium channels.
FIG. 28. Shows the response of HL5 cells as a function of the applied electric field
strength. Black points are the average of the response of four wells with no added compounds.
The solid line is a Boltzman fit to the data with E5Q=22 V/cm. The points are the screening
window: the difference between the response and the unstimulated response normalized to the
15 standard deviation of the response (see Appendix A3).
FIG. 29 The typical voltage response for CHO cells expressing a potassium channel and the
NaV3 sodium channel after a three separate stimulation cycles using surface electrodes.
FIG. 30 Shows the average ratiometric response of a population of cells grown in a 96 well
multiwell plate stimulated with monophasic stimuli of varying field strengths via surface electrodes.
20 The points in this curve are the average peak response of 4 stimulations on the same culture.
FIG. 31. Shows the cellular response as a function of the stimulus field for wild type
RBL. Error bars are standard deviations. Open symbols: no added blockers. Filled symbols: 400
(liM TEA added to block IRK1 channels.
Detailed Description of the Preferred Embodiment
25 Generally, the nomenclature used herein and many of the fluorescence, computer,
detection, chemistry and laboratory procedures described below are those well known and
commonly employed in the art. Standard techniques are usually used for chemical synthesis,
fluorescence, optics, molecular biology, computer software and integration. Generally, chemical
reactions, cell assays and enzymatic reactions are performed according to the manufacturer's
30 specifications where appropriate. The techniques and procedures are generally performed
according to conventional methods in the art and various general references, including those listed
below.
Lakowicz, J.R. Topics in Fluorescence Spectroscopy, (3 volumes) New York: Plenum
Press (1991), and Lakowicz, J. R. Emerging applications of fluorescence spectroscopy to cellular
35 imaging: lifetime imaging, metaUligand probes, multi-photon excitation and light quenching.
Scanning Microsc Suppl Vol. 10 (1996) pages 213-24, for fluorescence techniques.
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WO 02/08748 PCT/US01/21652
Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 n d ed. (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., for molecular biology methods.
Cells: A Laboratory Manual, 1 st edition (1998) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., for cell biology methods.
5 Optics Guide 5 Melles Griot® Irvine CA, Optical Waveguide Theory, Snyder & Love
published by Chapman & Hall for general optical methods.
Hille, B. Ionic Channels of Excitable membranes, Second Edition (1992) Sinauer
Associates, Inc., Sunderland, Mass. for general electrophysiological methods and properties of ion
channels.
10 Horowitz and Hill, The Art of Electronics, Second Edition (1989) Cambridge University
Press, Cambridge, U.K. for electronic circuits.
The following definitions are set forth to illustrate and define the meaning and scope of the
various terms used to describe the invention herein.
The term activation refers to the transition from a resting (non-conducting) state of an ion
15 channel to the activated (conducting) state.
The term activation threshold refers to the lowest potential above which measurable
opening of a channel occurs.
The term anode refers to an electrode when driven to a positive potential relative to earth
by an external source.
20 The term area of cellular stimulation means the area defined by two electrodes that
experiences significant electrical stimulation (typically 5V/cm or higher) in which the cells of
interest are located. Typically the area of cellular stimulation is larger than, or equal to, the area of
observation. For standard 96-well based measurements the area of cellular stimulation is typically
about 16 mm2.
25 The term area of observation means the portion of the system over which a measurement is
taken. The area of observation is typically an area of at least 0.5 mm2 for multiwell plate based
measurements.
The term bioluminescent protein refers to a protein capable of causing the emission of light
through the catalysis of a chemical reaction. The term includes proteins that catalyze
30 bioluminescent or chemiluminescent reactions, such as those causing the oxidation of luciferins.
The term bioluminescent protein includes not only bioluminescent proteins that occur naturally,
but also mutants that exhibit altered spectral or physical properties.
The term biphasic refers to a pulse with two parts, each with an opposite polarity.
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WO 02/08748 PCT/US01/21652
The term Boltzman function refers to the sigmoidal (i.e. step-like) response function
1 + exp
x — x
50
Ax
Where: y is the independent variable
y>0 is an adjustable parameter equal to the limit of the function as x — > oo
A is an adjustable parameter equal to step size
X50 is an adjustable parameter related to the midpoint of the step
Ax is an adjustable parameter describing the width of the step
The term cathode refers to an electrode when driven to a negative potential relative to earth
10 by an external source.
The term depolarize means to cause the transmembrane potential of a cell to become closer
to zero. In the case of cells that are normally at negative resting potentials, this term means that
the transmembrane potential changes in a positive direction.
The term effective concentration (50%) or EC50 refers to the concentration at which a
15 pharmacological compound has half the effectiveness compared to the maximal effectiveness at
high concentrations of the compound.
The term electrically excitable refers to a cell or tissue that responds to a suprathreshold
electrical stimulus by generating an action potential. Electrically excitable cells contain at least
one voltage-dependent ion channel type generating an inward current and at least one ion channel
20 type generating an outward current.
The term electrical stimulation means initiating a voltage change in cells using an
extracellular current pulse.
The term electrode means a controllable conductive interface between an instrument and a
test system.
25 The term electropermeablization refers to mild electroporation, in which the hydrated
pores created through the membrane are only large enough to pass water molecules and small
single-atom ions.
The term electroporation refers to a phenomenon in which the application of a large
electric potential across the membrane of a cell results in dielectric breakdown of the membrane,
30 and the creation of hydrated pathways through the membrane.
The term fluorescent component refers to a component capable of absorbing light and then
re-emitting at least some fraction of that energy as light over time. The term includes discrete
compounds, molecules, naturally fluorescent proteins and marco-molecular complexes or mixtures
of fluorescent and non-fluorescent compounds or molecules. The term fluorescent component also
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WO 02/08748 PCT/US01/21652
includes components that exhibit long lived fluorescence decay such as lanthanide ions and
lanthanide complexes with organic ligand sensitizers, that absorb light and then re-emit the energy
over milliseconds.
The term FRET refers to fluorescence resonance energy transfer. For the purposes of this
5 invention, FRET includes energy transfer processes that occur between two fluorescent
components, a fluorescent component and a non-fluorescent component, a luminescent component
and a fluorescent component and a luminescent component with a non-fluorescent component.
The term gene knockout as used herein, refers to the targeted disruption of a gene in vivo
with complete loss of function that has been achieved by any transgenic technology familiar to
10 those in the art. In one embodiment, transgenic animals having gene knockouts are those in which
the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered
non-functional by homologous recombination.
The term Hill function refers to the sigmoidal (i.e. step-like) response function
A
y( x )= yo
15 Where: y is the independent variable
yQ is an adjustable parameter equal to the limit of the function as x — > oo
A is an adjustable parameter equal to step size
*0 is an adjustable parameter related to the midpoint of the step
n is an adjustable parameter describing the steepness of the step
20
The term Hill coefficient refers to the parameter n in the Hill function.
The term hit refers to a test compound that shows desired properties in an assay.
The term homolog refers to two sequences or parts thereof, that are greater than, or equal
to 75% identical when optimally aligned using the ALIGN program. Homology or sequence
25 identity refers to the following. Two amino acid sequences are homologous if there is a partial or
complete identity between their sequences. For example, 85% homology means that 85% of the
amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in
either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5
or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein
30 sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are
homologous, as this term is used herein, if they have an alignment score of more than 5 (in
standard deviation units) using the program ALIGN with the mutation data matrix and a gap
penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, 1972,
volume 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this
35 volume, pp. 1-10.
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WO 02/08748 PCT/US01/21652
The term hyperpolarize means to cause the transmembrane potential of a cell to move
farther away from zero. In the case of cells that are normally at negative resting potentials, this
term means that the transmembrane potential changes in a negative direction.
The term inactivation means that an ion channel moves into the inactivated state.
5 The term inactivated refers to a voltage-dependent ion channel in a particular non-
conducting conformational state. Transitions into and out of the inactivated state are generally
slow relative to transitions between other conformational states. The inactivated state is usually
the preferred state at elevated transmembrane potentials. At low transmembrane potentials, the
inactivated state is unstable and relaxes to the resting state.
10 The term kernel means a mathematical function intended to be convoluted with one or
more other time-varying functions. In theory, the kernel can be any function that tends to zero as
the independent variable tends to ± oo. In practice, the kernel can be any waveform that can
programmed into an arbitrary wavefunction generator, or that can be generated by a computer-
controlled digital to analog (D/A) converter.
15 The term luminescent component refers to a component capable of absorbing energy, such
as electrical (e.g. Electro-luminescence), chemical (e.g. chemi-luminescence) or acoustic energy
and then emitting at least some fraction of that energy as light over time. The term component
includes discrete compounds, molecules, bioluminescent proteins and macro-molecular complexes
or mixtures of luminescent and non- luminescent compounds or molecules that act to cause the
20 emission of light.
The term transmembrane potential modulator refers to components capable of altering the
resting or stimulated transmembrane potential of a cellular or sub-cellular compartment. The term
includes discrete compounds, ion channels, receptors, pore forming proteins, or any combination of
these components.
25 The term membrane time constant or iy[ means the product of the membrane resistance
(R-M) and capacitance (Cm)-
The term monophasic refers to a pulse whose polarity does not change to the opposite
polarity.
The term naturally fluorescent protein refers to a protein capable of forming a highly
30 fluorescent, intrinsic chromophore either through the cyclization and oxidation of internal amino
acids within the protein or via the enzymatic addition of a fluorescent co-factor. The term includes
wild-type fluorescent proteins and engineered mutants that exhibit altered spectral or physical
properties. The term does not include proteins that exhibit weak fluorescence by virtue only of the
fluorescence contribution of non-modified tyrosine, tryptophan, histidine and phenylalanine groups
3 5 within the protein.
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The term naturally occurring refers to a component produced by cells in the absence of
artificial genetic or other modifications of those cells.
The term Multiwell plate refers to a two dimensional array of addressable wells located on
a substantially flat surface. Multiwell plates may comprise any number of discrete addressable
5 wells, and comprise addressable wells of any width or depth. Common examples of multiwell
plates include 96 well plates, 384 well plates and 3456 well Nanoplates™.
The term operably linked refers to a juxtaposition wherein the components so described are
in a relationship permitting them to function in their intended manner. A control sequence
operably linked to a coding sequence is ligated in such a way that expression of the coding
10 sequence is achieved under conditions compatible with the control sequences.
The term polarized cell means a cell with an electric potential difference across its cell
membrane.
The term rectification means that the conductance is non-linear, with a preferred direction.
The term release from inactivation refers to the conversion of an inactivated closed
15 channel, to a resting closed channel that is now capable of opening.
The term repetitive means to repeat at least twice.
The term repolarize means to cause the transmembrane potential of a cell to approach its
resting potential.
The term resting or resting state refers to a voltage-dependent ion channel that is closed,
20 but free from inactivation.
The term resting potential for a cell means the equilibrium transmembrane potential of a
cell when not subjected to external influences.
The term reversal potential for a particular ion refers to the transmembrane potential for
which the inward and outward fluxes of that ion are equal.
25 The term substantially parallel means that the distance between the surfaces of two objects
facing each other varies by less than 10 %, preferably less than 5 %, when measured at every point
on the relevant surface of each object.
The term targetable refers to a component that has the ability to be localized to a specific
location under certain conditions. For example, a protein that can exist at two or more locations
30 that has the ability to translocate to a defined site under some condition(s) is targetable to that site.
Common examples include the translocation of protein kinase C to the plasma membrane upon
cellular activation, and the binding of SH2 domain containing proteins to phosphorylated tyrosine
residues. The term includes components that are persistently associated with one specific location
or site, under most conditions.
35 The term threshold electroporation potential refers to the externally applied field strength
above which detectable electroporation of a living cell occurs.
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The term test compound refers to a chemical to be tested by one or more screening
method(s) of the invention as a putative modulator. A test compound can be any chemical, such as
an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a
combination thereof. Usually, various predetermined concentrations of test compounds are used
5 for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar. Test compound controls
can include the measurement of a signal in the absence of the test compound or comparison to a
compound known to modulate the target.
The term transformed refers to a cell into which (or into an ancestor of which) has been
introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid
10 molecule.
The term transgenic is used to describe an organism that includes exogenous genetic
material within all of its cells. The term includes any organism whose genome has been altered by
in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to
induce a specific gene knockout.
15 The term transgene refers any piece of DNA which is inserted by artifice into a cell, and
becomes part of the genome of the organism (i.e., either stably integrated or as a stable
extrachromosomal element) which develops from that cell. Such a transgene may include a gene
which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent
a gene homologous to an endogenous gene of the organism. Included within this definition is a
20 transgene created by the providing of an RNA sequence that is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention include DNA sequences that encode
the fluorescent or bioluminescent protein that may be expressed in a transgenic non-human animal.
The term transistor-transistor logic or TTL refers to an electronic logic system in which a
voltage around +5V is TRUE and a voltage around 0V is FALSE.
25 A uniform electric field means that the electric field varies by no more than 15 % from the
mean intensity within the area of observation at any one time.
The term voltage sensor includes FRET based voltage sensors, electrochromic
transmembrane potential dyes, transmembrane potential redistribution dyes, extracellular
electrodes, field effect transistors, radioactive ions, ion sensitive fluorescent or luminescent dyes,
30 and ion sensitive fluorescent or luminescent proteins, that are capable of providing an indication of
the transmembrane potential.
The following terms are used to describe the sequence relationships between two or more
polynucleotides: reference sequence, comparison window, sequence identity, percentage identical
to a sequence, and substantial identity. A reference sequence is a defined sequence used as a basis
35 for a sequence comparison; a reference sequence may be a subset of a larger sequence, for
example, as a segment of a full-length cDNA or gene sequence, or may comprise a complete
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cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length,
frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two
polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide
sequence) that is similar between the two polynucleotides, and (2) may further comprise a
5 sequence that is divergent between the two polynucleotides, sequence comparisons between two
(or more) polynucleotides are typically performed by comparing sequences of the two
polynucleotides over a comparison window to identify and compare local regions of sequence
similarity. A comparison window, as used herein, refers to a conceptual segment of at least 20
contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a
10 reference sequence of at least 20 contiguous nucleotides and wherein the portion of the
polynucleotide sequence in the comparison window may comprise additions or deletions (i.e.,
gaps) of 20 percent or less as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by the local homology algorithm
15 of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of
Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,
20 WI), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is selected. The term sequence
identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide
basis) over the window of comparison. The term percentage identical to a sequence is calculated
by comparing two optimally aligned sequences over the window of comparison, determining the
25 number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in
both sequences to yield the number of matched positions, dividing the number of matched
positions by the total number of positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence identity. The terms substantial
identity as used herein denotes a characteristic of a polynucleotide sequence, wherein the
30 polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at
least 50 to 60 percent sequence identity, more usually at least 60 percent sequence identity as
compared to a reference sequence over a comparison window of at least 20 nucleotide positions,
frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence
identity is calculated by comparing the reference sequence to the polynucleotide sequence which
35 may include deletions or additions which total 20 percent or less of the reference sequence over the
window of comparison.
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As applied to polypeptides, the term substantial identity means that two peptide sequences,
when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights,
share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, more
preferably at least 50 percent sequence identity, and most preferably at least 60 percent sequence
5 identity. Preferably, residue positions which are not identical differ by conservative amino acid
substitutions. Conservative amino acid substitutions refer to the interchangeability of residues
having similar side chains. For example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having amide-containing side chains is
10 asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and
histidine; and a group of amino acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine- valine, glutamic-aspartic, and asparagine-
1 5 glutamine.
Since the list of technical and scientific terms cannot be all encompassing, any undefined
terms shall be construed to have the same meaning as is commonly understood by one of skill in
the art to which this invention belongs. Furthermore, the singular forms a, an and the include plural
referents unless the context clearly dictates otherwise. For example, reference to a restriction
20 enzyme or a high fidelity enzyme may include mixtures of such enzymes and any other enzymes
fitting the stated criteria, or reference to the method includes reference to one or more methods for
obtaining cDNA sequences which will be known to those skilled in the art or will become known
to them upon reading this specification.
L Introduction
25 The present invention recognizes for the first time that the transmembrane potentials of
intact living cells comprising at least one voltage regulated ion channel, can be precisely
modulated via the application of repetitive electrical stimulation pulses to the fluid bathing the
cells. The present invention includes instrumentation and methods that provide for the accurate
and reliable modulation of the transmembrane potentials of intact living cells without significantly
3 0 disrupting their native cellular integrity.
As a non-limiting introduction to the breadth of the invention, the invention includes
several general and useful aspects, including:
1) Instrumentation including electrodes, and electrode arrays for reliably generating
uniform electrical fields in cultures of living cells in aqueous solution.
35 2) Multiwell plates comprising surface electrodes for high throughput and
miniaturized stimulation and analysis of ion channel or cellular activities.
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3) Systems for high throughput analysis of ion channel and cellular activities and for
use in drug discovery, analysis, screening and profiling.
4) Methods for modulating the transmembrane potential of a living cell via the use of
repetitive electrical stimulation.
5 5) Methods for screening the effects of test compounds on the activities of voltage
regulated, and non-voltage regulated ion channels, transporters and leak currents. Including
determining state-dependent pharmacological activity of compounds against ion channel and
transporter proteins.
6) Methods for profiling and selecting cells or clones based on their response to
10 electrical stimulation.
7) Methods for quantitative determination of cellular and ion channel parameters in a
high-throughput manner, and for quantification of the pharmacological effects of compounds on
those parameters.
8) Methods for the introduction of exogenous compounds into the intracellular spaces
15 of cells.
9) Methods for modulating the transmembrane potential of intracellular organelles,
and for screening test compounds against ion channels in these organelles.
10) Methods for characterizing the physiological effect of the transmembrane potential
on the function and regulation of physiological and biochemical responses, including gene
20 expression, enzyme function, protein activity and ligand binding.
11) Methods for programming or training adaptive neuronal networks or bio-
computers for specific functional or logical responses.
12) Methods for providing efficient neuronal interfaces for prosthetic devices
implanted into an animal, including a human.
25 These aspects of the invention and others described herein, can be achieved by using the
methods and instrumentation described herein. To gain a full appreciation of the scope of the
invention, it will be further recognized that various aspects of the invention can be combined to
make desirable embodiments of the invention. Such combinations result in particularly useful and
robust embodiments of the invention.
30 IL Electrodes and electrode arrays
In one embodiment, the present invention includes electrodes, and electrode arrays, for
creating electrical fields across the area of observation. Typically this is achieved via the use of a pair
of electrically conductive electrodes. An important design feature is that the electrode pairs create
well-defined electrical fields. Preferred electrode designs include electrode configurations that
35 maximize the electric field homogeneity experienced by the cells under observation.
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Generating uniform electric fields over the area of observation is important for electrical
stimulation for several reasons. Firstly, because the cellular response is sensitive to the magnitude
of the local electric field, non-uniform fields typically cause non-uniform responses in different
areas, leading to an increased scatter in the results. Secondly, the threshold for
5 electropermeablization is typically only a factor of 2-5 larger than the transmembrane potentials
required for electrical stimulation membrane (see Teissie and Rols, 1993, Biophys. J. 65:409-413).
Thus, if the electric field is too non-uniform, it may not be possible to stimulate all the cells in the
area of observation without also electropermeablizing some of them.
Field uniformity over a fixed area can be described in two ways: (1) the standard deviation
10 of the field magnitude divided by the average field magnitude in the area, and (2) the difference
between the highest and lowest field magnitudes, normalized to the average field magnitude in the
area.
a) Design of Electrodes
The simplest way to generate a uniform electric field in a conductive medium is to use two
15 identical, flat electrodes with surfaces that are aligned substantially parallel to each other.
Generally the closer the electrodes are to each other relative to their width in the transverse
direction, the greater the field uniformity will be. Typical round multiwell plate wells however
limit the width of electrodes that can be inserted into the wells, and also introduce two other effects
which reduce field uniformity.
20 The roundness of the wells provides a challenge to create a uniform field pointing in one
direction with two electrodes the width of the conductive saline between the electrodes is
constantly changing. Additionally the high surface tension of water generates variations in the
height of the saline across the well when dipper electrodes are inserted. The curved surface, or
meniscus, can perturb the electric field throughout the volume of the well. The depth of 100 juL of
25 saline in a 96-well plate is normally about 3.0 mm deep at the center and about 2.9 mm deep at the
edges of the well. When two stainless steel parallel plate electrodes are inserted, saline is drawn up
between the electrodes and the walls of the well causing depth variations over the area of
observation suggesting that the current paths throughout the volume of the saline curve around the
center, generating electric field non-uniformity.
30 In one aspect the present invention includes improved electrode designs, and systems for
electrical stimulation that address these issues to create substantially uniform electrical fields over
the area of observation.
In one embodiment, (FIG. 9A) the electrode pair comprises two substantially parallel
electrodes comprising an electrical insulator that is attached to the pair of electrodes to restrict
35 current flow to a defined region thereby creating a highly uniform electrical field.
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In another embodiment, (FIG. 9B) the electrode pair additionally comprises satellite
electrodes to create a more uniform electrical field.
In another embodiment, (FIG. 9D) the electrode pair is sub-divided into several pieces
separated by thin insulating dividers. In this case the potential applied to each electrode, expressed
5 as a fraction of the potential applied to the central most piece can be individually tuned to
maximize the field uniformity in the area of observation.
In another aspect, the present invention includes improved electrode designs (FIG. 9C)
that exhibit improved field uniformity over the area of observation via the elimination or reduction
of the meniscus effect.
10 In another aspect multiple electric potential sensors can be fabricated into the surface or
walls of the wells in a multiwell plate, or attached in arrays to the dipper electrode assembly.
These sensors can be monitored to manually or automatically adjust the individual electrodes, so as
to maximize field uniformity. This arrangement will be useful to allow a stimulating electrode
array to compensate for variations and imperfections in the well shape, volume of saline, variations
15 in the manufacturing process for the electrodes, damage to the electrode assembly, etc.
b) Placement of electrodes within the wells
For dipper electrodes, the ideal situation (in terms of creating a uniform electric field)
would be to have the bottoms of the electrodes touching the bottom of the well. This way, there
will be no fringing fields or field non-uniformity associated with vertical current paths. For a
20 removable structure, however, it is not desirable to require the electrodes to make contact with the
surface. Small deviations in the plate geometry can cause some electrodes to press into the
surface, causing damage either to the plate, the cells, or the electrodes. Additionally, in some
wells, the electrodes may not extend all the way to the surface. For these reasons it may be
desirable to design a small gap between the bottom of the electrode and the bottom of the well.
25 Accordingly in one aspect the present invention includes multiwell plates in which the area
of observation in the middle of the well is raised relative to area around the circumference of the
well, where the electrodes would be placed.
The fringing fields will cause non-uniformity over an inter-electrode distance roughly
equal to the gap between the bottom of the electrode and the bottom of the well. Therefore, this
30 gap should be kept as small as is practical, preferably in the range of 0.1 to 0.5 mm and the area of
observation should not typically include any part of the well within this distance from the
electrodes.
c) Manufacture of electrodes
Any electrically conductive material can be used as an electrode. Preferred electrode
35 materials have many of the following properties, (1) they do not corrode in saline, (2) they do not
produce or release toxic ions, (3) they are flexible and strong, (4) they are relatively inexpensive to
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fabricate, (5) they are non porous, and (6) they are easily cleaned. Preferred materials include
noble metals (including gold, platinum, and palladium), refractory metals (including titanium,
tungsten, molybdenum, and iridium), corrosion-resistant alloys (including stainless steel) and
carbon or other organic conductors (including graphite and polypyrrole). For many embodiments
5 stainless steel provides a preferred electrode material. This material is inexpensive, easy to
machine, and very inert in saline. Stainless steel oxidizes slowly to produce iron oxide when
passing current in saline, but this does not appear to affect the performance of the system. Iron
oxide has very low solubility in water and toxic levels of iron do not appear to be released.
Additionally any iron oxide deposits can easily be removed by soaking the electrodes in 10% nitric
10 acid in water for two hours, then rinsing thoroughly with distilled water.
Solid copper and silver electrodes may be used for some applications but are less preferred
for routine use because they corrode rapidly in saline. Gold plated copper electrodes are relatively
inert, but appear to lose their gold plating during prolonged electrical stimulation.
Electrolysis products can be contained or eliminated by coating the surfaces of the electrodes
15 with protective coatings, such as gelatin, polyacrilimide, or agarose gels. Another potentially useful
electrode material is an electrochemical half-cell, such as a silver/silver chloride electrode.
d) Electrode arrays
Dipper electrodes typically consist of one or more pairs of electrodes that are arranged in an
array that can be retractably moved into, and out of, one or more wells of a multiwell plate. Dipper
20 electrodes may be orientated into arrays that match the plate format and density, but can be in arrays
of any configuration or orientation. For example for a standard 96 well plate, a number of electrode
configurations are possible including electrode array arrangements to selectively excite one or more
columns, or rows, simultaneously.
An example of one embodiment of an electrode array of this type is shown in FIG. 1. In
25 this example, a 12 by 8 array of electrode pairs is formatted so as to fit into a standard 96-well
multiwell plate. In this case the electrodes (10) are approximately 4 mm wide, 1 cm long and 0.2
mm thick, and extend from a conductive comb (50) that is connected through a switch to one side
of the output stage of a high-power function generator. The electrodes are mounted parallel to
each other, 4 mm apart, with a non-conductive nylon spacer (20) in between. In this case, the
30 switch (330) enables one column of the 96 well plate to be selectively stimulated at a time,
however any temporal, or spatial, combination of stimulation protocols is potentially possible
given the appropriate configuration of switching, wiring and power function generator.
The entire array of electrodes is held in correct registration by a rigid non conductive member
(30) that keeps each electrode pair correctly spaced to accurately match a standard 96 well plate
35 layout. The non-conductive member (30) provides for the electrodes to move up or down while
precisely maintaining their registration with the multiwell plate.
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To provide for correct registration of the electrode array with a multiwell plate, the electrode
assembly can optionally comprise an outer border or flange (40) that can accommodate a standard 96-
well plate, and enables accurate plate registration. In some embodiments the border (40) can further
include a registration notch or indentation (80) to provide unambiguous plate registration.
5 In a preferred embodiment (Also shown in FIG. 1 A) the electrode array further comprises
means for retractably inserting the electrode array into the wells of the multiwell plate. In one
embodiment of this configuration, the electrode array further comprises an upper, movable support
member (90) to which the electrodes (10) are attached. The movable support member (90) is able
to move up or down relative to the non-conductive member (30) by sliding on four alignment pins
10 (70). Not shown in these figures is a spring that enables the movable support layer (90) to
automatically return to the upper position when downward force is no longer applied. A spacer
(60) provides the ability to lock the movable support layer (90) and electrodes (10) in the fully
lower orientation. This device allows the electrical stimulator to be used in manual and/or robotic
screening modes.
15 III. Multiwell plates for electrical stimulation
The multiwell plates of the present invention are designed primarily to provide for efficient
electrical stimulation of cells while at the same time enabling the optical analysis of
transmembrane potential changes. To accomplish this conductive surface electrodes may be
orientated in, or on, the walls, bottoms or lids of the multiwell plate.
20 In general such multiwell plates can have a footprint of any shape or size, such as square,
rectangular, circular, oblong, triangular, kidney, or other geometric or non-geometric shape. The
footprint can have a shape that is substantially similar to the footprint of existing multiwell plates,
such as the standard 96-well microtiter plate, whose footprint is approximately 85.5 mm in width
by 127.75 mm in length, or other sizes that represent a current or future industry standard (see T.
25 Astle, Standards in Robotics and Instrumentation, J. of Biomolecular Screening, Vol. 1 pages 163-
168,1996). Multiwell plates of the present invention having this footprint can be compatible with
robotics and instrumentation, such as multiwell plate translocators and readers as they are known
in the art.
Typically, wells will be arranged in two-dimensional linear arrays on the multiwell plate.
30 However, the wells can be provided in any type of array, such as geometric or non-geometric
arrays. The multiwell plate can comprise any number of wells. Larger numbers of wells or
increased well density can also be easily accommodated using the methods of the claimed
invention. Commonly used numbers of wells include 6, 12, 96, 384, 1536, 3456, and 9600.
Well volumes typically can vary depending on well depth and cross sectional area.
35 Preferably, the well volume is between about 0. 1 microliters and 500 microliters.
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Wells can be made in any cross sectional shape (in plan view) including, square, round,
hexagonal, other geometric or non-geometric shapes, and combinations (intra-well and inter-well)
thereof. Preferred are square or round wells, with flat bottoms.
The walls can be chamfered (e.g. having a draft angle). Preferably, the angle is between
5 about 1 and 10 degrees, more preferably between about 2 and 8 degrees, and most preferable
between about 3 and 5 degrees.
The wells can be placed in a configuration so that the well center-to well-center distance
can be between about 0.5 millimeters and about 100 millimeters. The wells can be placed in any
configuration, such as a linear-linear array, or geometric patterns, such as hexagonal patterns. The
10 well-to- well distance can be about 9 mm for a 96 well plate. Smaller well-center to well-center
distances are preferred for smaller volumes.
The wells can have a depth between about 0.5 and 100 millimeters. Preferably, the well
depth is between about 1 millimeter and 100 millimeters, more preferably between about 2
millimeters and 50 millimeters, and most preferably between about 3 millimeters and 20
15 millimeters.
The wells can have a diameter (when the wells are circular) or maximal diagonal distance
(when the wells are not circular) between about 0.2 and 100 millimeters. Preferably, the well
diameter is between about 0.5 and 100 millimeters, more preferably between about 1 and 50
millimeters, and most preferably, between about 2 and 20 millimeters.
20 The multiwell plate, will generally be composed of electrically non-conductive material
and can comprise an optically opaque material that can interfere with the transmission of radiation,
such as light, through the wall of a well or bottom of a well. Such optically opaque materials can
reduce the background associated with optical detection methods. Optically opaque materials can
be any known in the art or later developed, such as dyes, pigments or carbon black. The optically
25 opaque material can prevent radiation from passing from one well to another, to prevent cross-talk
between wells, so that the sensitivity and accuracy of the assay is increased. The optically opaque
material can also be reflective, such as those known in the art, such as thin metal layers, mirror
coatings, or mirror polish. Optically opaque materials can be coated onto any surface of the
multiwell plate, or be an integral part of the plate or bottom as they are manufactured. Optically
30 opaque material can prevent the transmittance of between about 100% to about 50% of incident
light, preferably between about 80% and greater than 95%, more preferably greater than 99%.
Since most measurements will not typically require light to pass through the wall of the
well, materials such as polymers can include pigments to darken well walls or absorb light. Such
application of pigments will help reduce background fluorescence. Pigments can be introduced by
35 any means known in the art, such as coating or mixing during the manufacture of the material or
multiwell plate. Pigment selection can be based on a mixture of pigments to dampen all
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background inherent to the polymer, or a single pigment or ensemble of pigments selected to filter
or absorb light at desired wavelengths. Pigments can include carbon black.
Surface electrodes can be embedded or otherwise attached to the wall in a variety of
formats and arrangements, for example as several narrow vertical electrode stripes. By
5 appropriately tuning the relative potentials of each stripe, uniform electric fields can be generated
in the area of observation. Further, using a circular insert, or by embedding vertical stripe
electrodes all around the well, uniform electrical fields can be generated in any direction across the
well. It would also be possible to create a uniform field in one direction, followed by a uniform
field in another direction. This could be useful for cell types whose electrical characteristics are
10 anisotropic, such as neural or muscle cells, or for cell types with large aspect ratios.
Each well also comprises a bottom having a high transmittance portion and having less
fluorescence than a polystyrene-bottom of at least about 90 percent of said bottom's thickness.
This property can be determined by comparing the fluorescence of an appropriate control bottom
material with the fluorescence of a test material. These procedures can be performed using well
15 known methods. Preferably, the bottom is a plate or film as these terms are known in the art. The
thickness of the bottom can vary depending on the overall properties required of the plate bottom
that may be dictated by a particular application. Such properties include the amount of intrinsic
fluorescence, rigidity, breaking strength, and manufacturing requirements relating to the material
used in the plate. Well bottom layers typically have a thickness between about 10 micrometers
20 and about 1000 micrometers. Preferably, the well bottom has a thickness between about 10
micrometers and 450 micrometers, more preferably between about 15 micrometers and 300
micrometers, and most preferably between about 20 micrometers and 100 micrometers.
The bottom of a well can have a high transmittance portion, typically meaning that either
all or a portion of the bottom of a well can transmit light. The bottom can have an optically opaque
25 portion and a high transmittance portion of any shape, such as circular, square, rectangular, kidney
shaped, polygonal, or other geometric or non-geometric shape or combinations thereof.
Preferably, the bottom of the multiwell plate can be substantially flat, e.g. having a surface
texture between about 0.001 mm and 2 mm, preferably between about 0.01 mm and 0.1 mm (see,
Surface Roughness, Waviness, and Lay, Am. Soc. of Mech. Eng. #ANSI ASME B46. 1-2985
30 (1986)). If the bottom is not substantially flat, then the optical quality of the bottom and wells can
decrease because of altered optical and physical properties of one or both.
For surface electrode embodiments, the bottom will preferably comprise strips of
electrically conductive material or coatings that overlap the edge of the wells of the multiwell plate
and are in electrical contact with the contents of the wells. The electrically conductive strips will
35 typically terminate at electrical connectors to enable facile attachment to the output stage of a high-
power function generator as described previously. The electrically conductive strips should have
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low enough resistance so that they can carry the stimulating currents without excessive loss in voltage
over their length. The resistance from the connector end to the farthest well end should be less than
10 and more preferably less than 1 £2, and more preferably still less than 0.1 £1 The cross-
sectional area of the electrically conductive strips should be large enough to accomplish the resistance
5 requirement For commonly employed electrical conductors, this cross sectional area should be at
least 10 _ 4 mm^ 5 and more preferable at least 10"3 rnrn^,
In practice, any conductive materials could be used as long as they are capped with a
conductive material that is inert in saline. Such materials include the noble metals (including gold,
platinum, and palladium) and the refractory metals (including chromium, molybdenum, iridium,
10 tungsten, tantalum, and titanium) as well as alloys thereof. Preferred materials for the conductive
material for surface electrodes include combinations of chromium, copper, gold, and indium-tin-oxide
that can be readily embedded or electroplated into or on the transparent bottom layer. Electrolysis
products can be contained or eliminated by coating the surfaces of the electrodes with protective
coatings, such as gelatin, polyacrilimide, or agarose gels.
15 Another potentially useful electrode material is an electrochemical half-cell, such as a
silver/silver chloride electrode.
The electrically conductive material coatings or surface modifications can be introduced
into the bottom using any suitable method known in the art, including vacuum deposition,
electroplating, printing, spraying, radiant energy, ionization techniques or dipping. Surface
20 modifications can also be introduced by appropriately derivatizing a polymer or other material,
such as glass or quartz, before, during, or after the multiwell plate is manufactured and by
including an appropriate derivatized polymer or other material in the bottom layer. The derivatized
polymer or other material can then be reacted with a chemical moiety that is used in an application
of the plate. Prior to reaction with a chemical moiety, such polymer or other material can then
25 provide either covalent or non-covalent attachment sites on the polymer or other material. Such
sites in or on the polymer or other material surface can be used to attach conductive layers to the
plates. Examples of derivatized polymers or other materials include those described by U.S. Patent
5,583,21 1 (Coassin et al.) and others known in the art or later developed.
Materials and manufacturing
30 The materials for manufacturing the multiwell plate will typically be polymeric, since
these materials lend themselves to mass manufacturing techniques. However, other materials can
be used to make the bottom of the multiwell plate, such as glass or quartz. The bottom can be
made of the same or different materials and the bottom can comprise polystyrene, or another
material. Preferably, polymers are selected that have low fluorescence and or high transmittance.
35 Polymeric materials can particularly facilitate plate manufacture by molding methods known in the
art and developed in the future, such as insert or injection molding.
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The multiwell plate of the present invention can be made of one or more pieces. For
example, the plate and bottom can be made as one discrete piece. Alternatively, the plate can be
one discrete piece, and the bottom can be a second discrete piece, which are combined to form a
multiwell plate. In this instance, the plate and bottom can be attached to each other by sealing
5 means, such as adhesives, sonic welding, heat welding, melting, insert injection molding or other
means known in the art or later developed. The plate and bottom can be made of the same or
different material. For example, the plate can be made of a polymer, and the bottom made of
polystyrene, cycloolefin, Aclar, glass, or quartz.
Miniaturized surface electrode designs are feasible in standard plate formats (96, 384, 1536)
10 as well as 3456 and higher plate densities. The throughput of such systems is potentially extremely
high. For example, assuming 3456 wells per plate screened at 30 plates per hour corresponds to an
overall throughput of approximately 800,000 wells per eight-hour day, which is approximately 8
times faster than is presently available, assuming equal plate read times.
An example of one embodiment of multiwell plate with surface electrodes is shown in
15 FIG. 2A. In this example, pairs of conductive strips (200) are attached in parallel to an optically
transparent bottom layer (210) such as glass, or plastic such as COC (see U.S. Patent Number
5,910,287, issued June 8, 1999) in a 96-well plate format. In this example, the strips of conductive
material (200) are approximately 2 mm wide, 10 urn thick, and separated by distance of
approximately 4 mm to enable optical analysis of the cells located in the wells (220), between the
20 electrodes through the optically transparent bottom layer (210). In other embodiments the strips of
conductive material can comprise stainless steel wires (from about 0.001 to about 0.010 diameter).
The optically transparent bottom layer (210) is attached to a 96-well multiwell plate array (230) and
replaces the normal plate bottom. The strips of electrically conductive material (200) overlap the
edge of the wells (220) of the 96-well multiwell plate and are in electrical contact with the contents of
25 the wells. The electrically conductive strips (200) terminate at electrical contacts (240) to enable
facile attachment to the output stage of a high-power function generator as described previously. In
this example, there are two electrode contacts per eight-well column in the first well of the column.
This permits the use of standard 96-well plate layouts, for simpler handling during cell culturing. No
cells or saline are inserted into these wells. This design permits the simultaneous stimulation of seven
30 wells in a single column. During the assay, the operator or a robot will temporarily attach wires to the
contacts, for example with push-pin test electrodes.
Another embodiment of a multwell plate with surface electrodes is shown in FIG. 2B. In
this embodiment, the transparent bottom layer (210) extends beyond the edge of the multiwell plate
(230). In this configuration, all wells remain available for use with cells and compounds. Further,
35 attachment of external wiring to the contacts (240) is simplified. Push-pin contacts, circuit-board
edge connectors, or zero- insertion force sockets can be used to make contact with the electrodes. The
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extended bottom layer (210) may make the plates less convenient to manipulate during routine use.
This can be remedied by bringing the electrode traces (200) to the reverse side of the bottom layer
(210) during the manufacturing process. This can be accomplished by several methods. For
example, using two-sided processing of the plates to create contact areas, through-holes can be made
5 and electroplated, or conducting traces can be wrapped around the edge of the bottom layer. As
another example, the bottom layer can be made of a flexible insulating material. Then, after making
the structure as shown in FIG. 2B 5 the part of the bottom layer which protrudes from the edge of the
plate can be folded and attached to the underside of the plate.
Another embodiment of a multwell plate with surface electrodes is shown in FIG. 2C. In
10 this embodiment, the electrodes (200) are attached to the contact pads (240) with narrow via wires
(205). This permits the use of standard 96-well plate layouts, for simpler handling during cell
culturing. In this embodiment, all of the electrodes of one polarity are shorted together. Selection of
a single column is accomplished by supplying the current pulse to only one electrode of the other
polarity. In this embodiment, no cells, saline, or compounds are placed into the final column where
1 5 the contact pads are. During the assay, the operator or a robot will temporarily attach wires to the
contacts, for example with push-pin test electrodes.
Another embodiment of a multwell plate with surface electrodes is shown in FIG. 2D. In
this embodiment, the electrodes (200) are aligned parallel to the longer dimension of the 96-well
plate. This design is essentially similar to the design shown in FIG. 2A, with the exception that
20 eleven wells in a row will be simultaneously stimulated.
Preferred materials for the conductive material for surface electrodes include combinations of
chromium, copper, gold, and indium-tin-oxide that can be readily embedded, attached, or
electroplated into or on the transparent bottom layer. In practice, any conductive materials could be
used as long as they are capped with a conductive material that is inert in saline. Such inert materials
25 include the noble metals (including gold, platinum, and palladium), the refractory metals (including
chromium, molybdenum, iridium, tungsten, tantalum, and titanium), corrosion-resistant alloys
(including stainless steel), and carbon or other organic conductors (including graphite and
polypyrrole) as well as combinations or alloys of these materials.
IV. Systems for Electrical Stimulation and Spectroscopic Measurement
30 The present invention includes systems for automated electrical stimulation and
spectroscopic measurement, comprising: at least one electrode assembly, a means for electrical
stimulation, an optical detector, and computer control means to coordinate the generation of
electrical stimuli, collection of data and movement of multiwell plates. The system can further
comprise means for fluid addition. In one aspect these systems are designed for modulating,
35 characterizing and assaying the activity of ion channels, transporters, leak currents present in or on
the surfaces of living cells, and for rapidly screening for the effects of test compounds on the
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effects of channel or cellular activities. The present invention is also directed to chemical entities
and information (e.g., modulators or chemical or biological activities of chemicals) generated or
discovered by operation of workstations of the present invention.
FIG. 3 shows a block diagram of the major electrical and optical components for one
5 embodiment of a system for automated electrical stimulation and spectroscopic measurement. In
this example a 96-well multiwell plate dipper electrode array (FIG. 1) was used for electrical
stimulation. In addition to the stimulator electrode array, the system has several additional
electrical, optical and mechanical components, as described in detail in commonly owned U.S.
Patent Application No. 09/1 18,728, filed July 24, 1998.
10 In this embodiment, a National Instruments (Austin, TX) PC-DIO 24 digital input/output
card on board the computer (300) is used to set the proper channel on a l-to-12 switch (330)
(National Instruments ER-16). The computer controlling the fluorescent plate reader (300) also
sends out a TTL signal to trigger the function generators (310) when the stimulus is programmed to
begin. Stimulus signals are generated by two arbitrary waveform generators (310). The function
15 generators are Tektronix (Beaverton, OR) model number AFG310. The first triggers a series of
TTL pulses to the second which is programmed with the individual stimulus waveform. More
complex waveform trains can be generated by connecting multiple waveform generators in series
and/or in parallel. These waveform generators would be triggered by the computer-generated TTL
pulse or by each other. Alternatively, an A/D converter or a sound card on board the computer
20 could be used to generate a train of stimuli. In this case, commercially-available or custom
software could be used to program the waveform train, or to change the waveform during the train.
The train of stimuli is sent through a high-power amplifier (320), through the switch
(330), and into the stimulator head (370). In this case the amplifier was built using the APEX
PA93 chip (Apex Microtechnology Corp, Tucson, AZ) following a circuit provided by the
25 manufacturer. Preferred amplifiers for the present application would typically meet, or exceed the
following specifications: ±100V DC in, 100 GQ, input impedance, 20X voltage gain, ±90V out, ±3
A out, 10 Q, output impedance.
The majority of current passes through the saline between the electrodes, typically in a
single eight-well column of the microtiter plate (350) at a time. Excitation light at 400 ± 7.5 nm
30 illuminates the stained cells from below, and emitted fluorescent light is measured at two
wavelengths via the detector module (340) blue at 460 +/- 20 nm and orange at 580 +/- 30 nm; (see
Gonzalez et aL, Drug Discovery Today 4: 431-439, 1999). Once a column of cells has been
stimulated the computer (300) triggers the motor (360) to move the multiwell plate (350) to a new
position ready for the next stimulation.
35 For a typical 96-well multiwell plate, the electrodes are 4 mm wide with a gap (g) of 4
mm. Stimulation is usually performed in a volume of 100 \xL of physiological saline in the well.
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With this volume of saline, the depth averages approximately 3.0 mm (this depth varies by as
much as 20% across the well due to the meniscus effect). The electrodes rest approximately 0.5
mm off the bottom of the wells. The electric field (E) applied across the cells is estimated as the
voltage across the electrodes (Vq) divided by the electrode gap (g), E = V Q /g . This is an
5 overestimate of the actual field because of the influence of electrochemical reactions at each
electrode which consume approximately 1.5 V. In the typical voltage ranges used for stimulation
(10 to 60 V/cm), this overestimate is on the order of approximately 10%. Accurate measurement
and calibration of the field can be performed by mapping the electric potential in the well when
current is passed.
10 The present invention also includes automated workstations that are programmably
controlled to minimize processing times at each workstation and that can be integrated to minimize
the processing time of the liquid samples for electrical stimulation and analysis.
Typically, a system of the present invention would include one or more of the following:
A) a storage and retrieval module comprising storage locations for storing a plurality of chemicals
15 in solution in addressable chemical wells, a chemical well retriever and having programmable
selection and retrieval of the addressable chemical wells and having a storage capacity for at least
1 00,000 addressable wells, B) a sample distribution module comprising a liquid handler to aspirate
or dispense solutions from selected addressable chemical wells, the chemical distribution module
having programmable selection of, and aspiration from, the selected addressable chemical wells
20 and programmable dispensation into selected addressable sample wells (including dispensation
into arrays of addressable wells with different densities of addressable wells per centimeter
squared), C) a sample transporter to transport the selected addressable chemical wells to the sample
distribution module and optionally having programmable control of transport of the selected
addressable chemical wells (including adaptive routing and parallel processing), D) a system for
25 automated washing, staining, and timed incubation of cells in multiwell plates, E) a system for
automatically transporting cell plates and test compound plates between the various workstations,
F) a system for automated electrical stimulation and spectroscopic measurement, and a data
processing and integration module, G) a master control system which co-ordinates the activities of
any of the above subsystems.
30 The storage and retrieval module, the sample distribution module, and the system for
automated electrical stimulation and spectroscopic measurement are integrated and programmably
controlled by the data processing and integration module. The storage and retrieval module, the
sample distribution module, the sample transporter, the system for automated electrical stimulation
and spectroscopic measurement and the data processing and integration module are operably linked
35 to facilitate rapid processing of the addressable sample wells. Typically, devices of the invention
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can process at least 100,000 addressable wells in 24 hours. This type of system is described in
U.S. Patent No. 5,985,214, issued 11/16/99.
Microfluidic Systems
The present invention also includes the use of electrodes that have been incorporated into
5 microfluidic chips and which provide for highly miniaturized electrical stimulation and analysis.
Such systems include those, for example, described in U.S. Patent No., 5,800,690 issued
September 1, 1998 to Chow et al, European patent application EP 0 810 438 A2 filed May 5 1997,
by Pelc et ah and PCT application WO 98/00231 filed 24 June 1997 by Parce et ah These systems
typically use electrogenic fluid movement to manipulate small fluid volumes within
10 microcapillaries present on glass or silicon chips. These microfluidic chip based analysis systems
can provide massively parallel miniaturized analysis. Such systems are preferred systems of
spectroscopic measurements in some instances that require miniaturized analysis.
For example, the microfabricated fluorescence-activated cell sorter described by Fu et ah
(Nature Biotechnology J/7: 1109-11, 1999) could be easily modified to have a pair of electrodes
15 placed in, or near the optical interrogation region. Using the methods described herein, individual
cells could be electrically stimulated and individually sorted based on their response to the
stimulation. This method would greatly simplify the process of obtaining stable clones containing
the desired expression of channels. In another aspect, screening of test compounds on single cells
could be performed with a microfluidic device equipped with one or more additional fluid injection
20 ports and one or more embedded electrical stimulator devices built and operated based on the
methods described herein.
V. Electrical Stimulation Methods
a) Introduction
Without being bound to any mechanism of action, the present inventors provide the
25 following description for the effect of electrical stimulation on cellular transmembrane potentials.
Typical voltage-dependent ion channels have a variety of conducting and non-conducting
states that are regulated by the local relative transmembrane potential of the cell. By appropriately
applying external electrical fields to the cells, portions of the cell membrane can be driven to any
desired transmembrane potential, thereby enabling the regulation of the conduction states of
30 voltage dependent ion channels present within the cell. If the applied electrical field is
appropriately varied, it is possible to sample a number of conductance states of most ion channels,
thereby cycling them through resting, activated, and inactivated states.
Depending on the ion channel in question, activation of the ion channel can lead to the
release, or uptake, of ions into the cell that can result in global transmembrane potential changes in
35 the cell. By applying a repetitive train of electrical stimuli, separated by a time interval smaller
than the membrane time constant, large sustained membrane voltage changes can be created via a
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stepwise accumulation or loss of ions. This process allows the direct measurement of many ion
channels and provides a facile method whereby the transmembrane potential of the cell can be
externally controlled. This approach therefore provides for improved methods of drug discovery
that are compatible with high throughput screening.
5 b) Overview of a typical stimulation protocol
The simulated influence of a typical biphasic electrical stimulation protocol on a cell line
expressing a voltage activated sodium channel is illustrated, in simplified form, below. The
following description assumes that the cell line has no significant expression of other ion channels,
and that the resting transmembrane potential of the cell is above the threshold for inactivation of
1 0 the sodium channel in question. In FIG. 4, the upper panel shows the time course of the applied
electrical field (E), the middle panel shows the simulated inward sodium currents (iNa) * n response
to the applied electrical field, and the lower panel shows the idealized average transmembrane
potential of the cell (V m ). In this example, the recordings relate to the changes in these parameters
that a single cell placed in the center of the applied electrical field would be typically expected to
1 5 experience during an electrical stimulation wave train.
Referring to the first pulse, establishing the first electrical field causes a potential drop
across the cell that is maximal, with respect to the resting transmembrane potential of the cell, at
the edges of the cell closest to the electrodes (see Hibino et aL, Biophysical Journal 64:1789-1800,
1993; Gross et al. 1986, Biophys. J. 50:339-348). The magnitude of the electric field-induced
20 transmembrane potential change AV m at a given point of the membrane in an idealized spherical
cell can be described by the formula (Ehrenberg et al., Biophys. J. 51:833-837, 1987):
AV m =-fgrEcos&.
(1)
In Equation 1,/is a factor dependent upon the conductivity of the membrane, g is a geometric
25 factor of order 1, r is half the diameter of the cell parallel to the electric field, E is the local
magnitude of the electric field, and 6 is the angle between the local direction of the field and a line
drawn from the center of the cell to the point of the surface being considered. For most intact
mammalian cells, in which the membrane conductivity is very low compared to the conductivity of
the solution bathing the cells, the factor /« 1. In practice, cells are rarely spherical when attached
30 to a substrate and an accurate estimate of the actual magnitude of the electrical field induced
transmembrane potential changes may be empirically determined.
As a result of the applied electrical field, the membrane on the side nearest to the anode is
driven negative, while the membrane on the side nearest the cathode is driven positive. In cells in
which one edge is driven sufficiently negative to locally lower the transmembrane potential below
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the threshold potential for release of inactivation for the ion channel in question, the applied
electrical field causes the sodium channels located on this edge to enter the resting state. On the
other side of the cell, the transmembrane potential is driven positive of the resting potential.
Because the resting transmembrane potential of the cell is assumed to be above the threshold for
5 inactivation, sodium channels on this side of the cell remain inactivated and do not pass current. If
the resting transmembrane potential were instead below the inactivation threshold, channels on this
side of the cell would activate and pass current.
When the applied field is reversed, the profile of transmembrane potential changes also
reverses. The transmembrane potential changes induced by the electric field on the patches of
10 membrane at the extreme edges of the cells switches polarity. The channels on the side that was
driven negative during the first phase of stimulation are now driven positive. If the stimulation
parameters are properly chosen, these channels are now driven above the activation potential and
begin to allow sodium ion influx. This is shown in FIG. 4, as the first smaller peak of sodium
influx into the cell. The sodium channels rapidly inactivate after a characteristic time. Meanwhile,
15 on the other side of the cell, the transmembrane potential is driven negative so that the sodium
channels release from inactivation and move into the resting state.
When the second stimulus phase ends, all parts of the membrane rapidly return to a new
average transmembrane potential. If the average transmembrane potential is now above the
activation potential of the sodium channels, the channels on the side of the cell that was driven
20 negative during the second phase of stimulation activate and begin to allow sodium ion influx.
This is shown in FIG. 4, as the second larger peak of sodium influx into the cell. The sodium
channels rapidly inactivate after a characteristic time. In this case sodium influx is typically larger
from the second side than the first side, since the driving force for sodium entry is larger when this
part of the membrane is driven more positive by an electric field.
25 Each pulse of sodium channel influx raises the average transmembrane potential of the cell
(FIG. 4, lower panel). This rise in transmembrane potential can be detected by any of the methods
described herein, but is conveniently measured via fluorescence emission ratio changes of a FRET
based voltage-sensitive dye. Due to leakage currents present in all cells, this average
transmembrane potential shift decays exponentially to the original resting transmembrane
30 potential. The time dependency of this response, the membrane time constant (x m ), depends upon
the membrane capacitance and membrane resistance, and is highly variable from one cell type to
another. For example, time constants can vary from 100 |ns to over one second, depending on the
cell type. Typically the membrane time constant is around 100 ms for most engineered cell lines.
To provide a net accumulation of sodium influx the stimulus pulse is repeated before the
35 transmembrane potential has time to decay to the resting transmembrane potential. During
subsequent rounds of electrical stimulation, positive charge is steadily accumulated into the cell
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raising the average transmembrane potential in an approximately stepwise fashion with each
repetition of electrical stimulation. After each pulse of electrical stimulation, the magnitude of the
sodium ion influxes become steadily smaller as the average transmembrane potential approaches
the sodium ion reversal potential. Eventually an equilibrium transmembrane potential is
5 established in which leakage of current out of the cell equals the current influx due to electrical
stimulation.
c) Adjustable parameters for the stimulus waveforms
The present invention includes the use of any waveform kernel with any repetition
procedure capable of selectively activating ion channels in living cells. The kernel is the
10 repeatable structure that forms the basis of the stimulus train. In FIG. 4, the kernel is a biphasic
square pulse, but in principle it could be any limited-time wave function. The time duration of the
kernel sets the maximum rate at which it can be repeated. The repetition procedure dictates how
and when the kernel is presented to the sample. In FIG* 4, the repetition rate is fixed and continues
for a total of ten cycles. However the repetition rate need not be fixed.
1 5 Additionally, the kernel can be changed during the stimulus train, so that each time the
repetition procedure calls for a stimulus pulse, a different wave function could be used.
Furthermore, a feedback mechanism could be used to alter the kernel and/or the repetition
procedure based upon the measured response of the system.
The use of arbitrary waveform generators to create the stimulus kernels and trains allows
20 for a virtually unlimited variation in the waveform in order to tune the electrical stimulus to a
particular cell type or specific ion channel. The pulse train can be readily modulated via the
variation of a number of separately controllable components.
1 . The shape of the individual pulses.
The waveform kernel that is repeated during the stimulus train can be changed with nearly
25 endless permutations using a arbitrary digital waveform generator, such as Tektronix AFG 310.
FIG. 5 shows a schematic representation of a biphasic square waveform to illustrate some of the
variables that can be modulated. In FIG. 5, the pulse train consists of a starting field E\ (400), that
lasts for a time t\ 9 a rapid increase in potential (410), that takes a time t2, until reaching a first
stimulating field E2 (420) that lasts for time t3, a rapid decrease in potential (430) that takes time
30 t4, until reaching a second stimulating field (440), E3 that lasts a time ts, a rapid increase in
potential (460) that takes time t6, until reaching the finishing field (470), E4 that lasts a time tj
until the cycle is repeated. The magnitude and polarity of the electrical fields to E4 are
separately controllable and may be both statically and dynamically varied as described below. The
times for which the electrical potentials are applied to the cells, times t\ 9 t3, t5, and tj are also
35 separately controllable and may be both statically and dynamically varied between 0 and 10 s
during a wave train, as described below. Finally the changes in potential that occur over times t2,
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t4 and tg , may occur over variable time periods between 0 and 100 ms and be either linear or non
linear to create waveforms of variable shapes.
Some examples of these types of variation in the waveform are shown in FIG. 6 (a)
Biphasic waveform, as shown in FIG. 5, repeated at a rate f. (b) A modified biphasic waveform.
5 A short interval has been added between the stimulation phases of the wave train. This allows
current to flow through the channels released from inactivation during the first pulse, (c)
Monophasic waveform. Only channels on the side of the cell facing the anode will be released
from inactivation. (d) A ramped waveform. The anode-facing channels will be released from
inactivation by the square wave. The channels will activate and pass current during the ramp. The
10 ramp allows the channels to open and pass current at more negative local potentials, so that even
when the cell is near the reversal potential for sodium ions, large currents can still flow. The point
along the ramp at which the channels will open varies, (e) A biphasic triangular or sawtooth
waveform. Ramping may allow the voltage-dependent transitions between states to occur more
uniformly as the global membrane potential changes. Monophasic triangular waveforms are also
15 possible, (f) A sinusoidal waveform. This type of waveform may reduce electrical noise during
high frequency stimulation, (g) A short burst of sinusoidal waveforms, (h) Bursts of sinusoidal
waveforms, each with different fundamental frequency. This type of stimulation may prove useful
for studying plasticity effects. The first burst(s) are used to train the system or begin a process,
while the subsequent bursts(s) are used to assay the system.
20 Variations in waveform shape may be useful in maintaining fixed stimulus conditions
during the pulse train. For example, the transmembrane potential excursions experienced by a
highly polarized cell will vary as its average transmembrane potential changes from around —90
mV at the beginning of the stimulation cycle to around +60 mV after several repetitive stimulation
cycles. As a consequence, the applied electrical field required to efficiently release an ion channel
25 from inactivation varies as the average potential of the cell varies during the course of several
stimulation cycles. To take this effect into consideration it may be useful, under certain
circumstances, to change the relative balance between the positive (E2) and negative (E3) phases
of stimulation as the wave-train progresses.
Some cell lines, for example HEK-293, have a resting average transmembrane potential
30 below the activation threshold of some voltage-activated sodium channels. In these cells as the
transmembrane potential rises during stimulation as a result of sodium ion influx, the sodium
channels can open independently of the applied electrical stimulation. This can be improved by
using a sloped current pulse (i.e by increasing t2 and t4). Then, the channels can pass current for a
defined time just above the activation voltage, independent of the average transmembrane potential
35 of the cell.
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2. The overall amplitude of the individual pulse (E 2 and E 3).
The magnitude and polarity of the pulse amplitude controls the relative transmembrane
potential excursions experienced by the cell during a stimulus pulse. Pulse amplitudes can be
altered for the entire train, or for the individual pulses to accommodate different channels and cell
5 types, as discussed in more detail below. In general, the magnitudes of E2 and E3 are selected to
ensure that the ion channel of interest is efficiently activated, and released from inactivation during
each stimulation cycle, while at the same time not of sufficient magnitude so as to cause
irreversible electroporation of the cells. Preferred pulse amplitudes for E2 and E3 are typically in
the range of 5 to 60 V/cm for most ion channels when expressed in non-excitable mammalian cells
10 with average sizes from 10 to 25 (im, and may vary either positive or negative relative to earth. As
above, the amplitude of the stimulus can be changed during the pulse train to maintain stable
stimulus conditions as the average transmembrane potential changes. Preferred pulse amplitudes
are inversely dependent upon average cell size. So, the technique can also be used on cells which
are smaller or larger than 1 0 to 25 |am, by altering the pulse amplitude.
1 5 3. The duration of the individual pulses and t O.
Many channels require alterations in the transmembrane potential for extended periods of
time to release them from inactivation, prior to opening. For example, many voltage-dependent
sodium channels generally need to experience a transmembrane potential below -90 mV for
several milliseconds before they are released from inactivation. Efficient use of the electrical
20 stimulation protocol therefore typically requires that the duration of the pulses t3 and t5 are
sufficient to enable complete, or almost complete, release from inactivation for the ion channel of
interest. In some cases it may be desirable to tune the magnitude of t3 and t5 to enable the
selective release from inactivation of one class, but not another class of ion channel in a cell that
expresses several ion channel types. In other cases it may be desirable to make t3 and t5 very
25 small to achieve low levels of release from inactivation for the channels. Typically the preferred
pulse duration is matched to the characteristic time for transitions between the desired voltage-
dependent states for the ion channel of interest, and these are typically in the range of about 0.1 to
100 msec for most ion channels.
To avoid excessive electrolysis of water and consequent gas bubble generation, the
30 duration of the pulses t3 and t5 should be kept as short as possible, while still achieving the desired
electrical stimulation. Water electrolysis at a metal/water interface typically occurs when the
magnitude of the voltage difference between the metal and the water exceeds about 0.8 V. In some
cases, the stimulus parameters required to produce cellular stimulation also cause water
electrolysis. Some generation of gas at the electrodes is typically acceptable as long as the charge
35 per unit area of the electrode/water interface delivered during any single polarity phase of a single
pulse is less than about 100 jnC/mm^. Exceeding this limit typically causes gas evolution and
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bubble formation that significantly affects field uniformity. The presence of bubbles on the
electrode surface occludes that part of the electrode, and can cause alterations in the electric field
uniformity. Generation of large amounts of gas can also cause oxidative damage to the cells and
the dyes in the well.
5 In a 96- well plate with 100 jiL of physiological saline with resistivity 70 Q-cm, the
resistance of the saline between two parallel plate electrodes with a 4 mm gap between them
inserted into the well to within 0.5 mm of the bottom of the well, is approximately 230 Q.. Each
electrode has a contact area with the saline of about 24 mm^ Thus, any single-polarity phase of
the stimulus protocol should not deliver more than about 2.4 mC of charge. A voltage difference
10 of about 10 V applied between the plates generates an electric field of about 25 V/cm in the saline.
This voltage will draw about 43 mA of current. Thus for this electrode configuration, a square
wave, single-polarity pulse should not exceed about 55 milliseconds in duration in order to limit
the charge to less than 2.4mC.
4. The gap between successive stimuli (t \ and t j).
15 Changing the value of t\ and tj globally for the train, or adjusting it for each individual
pulse during the train, is useful for optimizing the stimulation protocol for specific ion channels.
Additionally the approach is also useful for determining certain cellular and channel properties
including the open channel time and the time course of the channel activation and inactivation.
For example, for assays involving voltage regulated sodium channels, the insertion of a
20 time delay (t\+tj) between pulses equal to, or less than, the average sodium channel open time
allows for a quantitative measurement of the inactivation kinetics of the channel. The inactivation
kinetics are directly related to the average open channel time. Thus, assays using short interpulse
intervals allows for the detection of compounds whose primary effect is on inactivation kinetics, a
mechanism which is otherwise inaccessible using high-throughput techniques.
25 In most cases the time delay between successive stimuli would be less that the membrane
time constant in order to obtained sustained increases in transmembrane potential. Typically
optimal frequencies of stimulation if) are within the range x]yfl - / ^ x b~* where xm is the time
constant for decay of transmembrane potential changes, and xb * s the average channel open time.
Some channels do not inactivate, and for these cells the stimulation frequency may be determined
30 empirically. Additionally, the stimulation frequency / cannot exceed the inverse of the time
duration of the stimulus kernel.
Additionally, for certain cell types, it may prove desirable to stimulate at a slower rate.
For example, slower stimulation rates may be preferred for cells with high channel densities, or for
assays in which higher pharmacological sensitivity is required. Alternatively for these cases, a
35 monopolar stimulus could be used. This would only release from inactivation the sodium channels
on one side of the cell, but the maximum frequency of stimulation could be doubled.
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5. The duration of the train of pulses, or number of pulses in the train.
Cellular and channel properties can be assayed both in dynamic (i.e. rise and fall times,
alterations in response shape, etc.) and static modes. Both modes require stimulus train durations
long enough to explore all the events of interest, yet not longer than necessary to complete the
5 assay. Typical stimulation times comprise 10 msec pulses, at 25 V/cm pulses repeated at a
frequency of 20 Hz for 3 seconds. Adjusting these parameters allows assay times to be reduced, or
to explore processes with both fast and slow time scales.
6. Multiple pulse trains.
In some cases it is useful to repeat pulse trains, or to perform a measurement on the same
10 cells with two different pulse trains. One example would be to completely characterize the
properties of a channel by measuring the response as a function of stimulus frequency and
duration, using a single plate of cells subjected to multiple stimulus trains. Another example
would be to examine plasticity of the response (i.e. activity-dependent changes in response). One
or more stimulus trains would condition the response, while sets of measurement trains before and
15 after the conditioning would determine the changes due to activity.
Feedback of stimulus parameters based upon dynamic measurements of the response.
The present invention can also be used to create a voltage clamp device, by using a
dynamic feedback loop to maintain the average transmembrane potential at a preset value. By
measuring the transmembrane potential using a fast fluorescent output as described below, then
20 changing stimulus parameters to compensate for any changes in transmembrane potential, it is
possible to dynamically control the transmembrane potential of the cells. The current necessary to
maintain that potential would then be determined by computer control of the stimulus parameters.
The use of high frequency stimulation to avoid electrolysis
During typical stimulation parameters, a peak current of approximately 50 mA passes
25 through the solution between the electrodes. During this time various electrochemical reactions
occur which typically generate toxic species to the cells. Preliminary experiments have shown that
most mammalian cells typically respond normally for approximately two minutes of electrical
stimulation using stainless steel electrodes. However prolonged stimulation for longer time periods
appears to lead to a loss in cell health and viability. At sufficiently high pulse frequencies, such
30 that the metal-saline interface does not reach the potential for electrolysis of water (approximately
±1V for stainless steel in saline), current can be passed capacitively and no toxic products will be
generated. In the electrical stimulator shown in FIG. 1, in which each electrode has an area of
about 24 mm 2 in contact with the saline, the capacitance per electrode is around 1-10 jiF
(Robinson, 1968, Proc. IEEE 56:1065-1071). At 50 mA, this capacitance charges to 1 V in around
35 20-200 |is. This is at the lower limit of the useful pulse duration times.
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WO 02/08748 PCT/US01/21652
Alternatively it is possible to perform electrical stimulation without generating electrolytic
products. Several treatments are available which can increase the capacitance of the metal-saline
interface by factors of 2-100. These include surface roughening, electroplating with platinum
black or gold black, and deposition and activation of iridium/iridium oxide, titanium/titanium
5 nitride, or polypyrrole films. Using stimulation parameters, which avoid irreversible
electrochemistry, these surface treatments do not degrade when passing current.
VI. Expression of Ion Channels
a) Selection of the cell type
The present invention can be used with any type of cell, including animal cells, plant cells,
10 insect cells, bacterial cells, yeast and mammalian cells. For screening for human therapeutics
mammalian cell lines are preferred, such cell lines include tissue culture cell lines that can be
relatively easily grown, and can be readily transfected with high efficiency. Many tissue cell lines
are commercially available through the American type culture collection (ATCC) see
( http://www.atcc.orgX as well as the European collection of cell cultures (ECACC)
15 ( http://www.camr.org.uk\
Additionally in some cases primary cell lines, or tissue slices may also be preferred for
screening when it is required to express, or measure, the response of the ion channel of interest in
its native physiological context. This approach may be useful either as a primary or a secondary
screen to screen for specificity, selectivity or toxicity of candidate therapeutics, and is discussed in
20 detail in section X.
For assays performed on cultured cell lines, the main selection criteria are the resting
transmembrane potential of the cell line, and the presence of endogenously expressed ion channels.
The selection of appropriate cell lines for specific ion channels of interest are dependent on the
voltage dependent properties and ion selectivity of the ion channel of interest. These
25 considerations are reviewed in detail for a number of ion channels in section VIII, Stimulation
Protocols.
In some cases it is desirable to use a cell line which has no (or very low) detectable
endogenous expression of other ion channels. Cells of this type include CHO-K1, CHL, and
LTK(-) cells. These cells inherently have a resting potential in the range of +10 to -30 mV, which
30 is above the activation and inactivation thresholds of most voltage-dependent channels. Use of
these cell lines has the advantage that the ion channel of interest is the major modulator of
transmembrane potential within the cells so that screening assay data are generally easily and
unambiguously interpreted.
In some cases the use of a cell line with no other ion channels may not be practical to
35 create a workable assay. For example, it may be necessary to maintain a voltage-regulated ion at a
highly polarized transmembrane potential. In this case it is necessary control the transmembrane
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WO 02/08748 PCT/US01/21652
potential via the expression of a second ion channel. For example to assay a rat brain type Ha
sodium channel in the resting state requires the transmembrane potential to be maintained below
the threshold activation potential of the sodium channel, in this case around —60 mV. To achieve
this it is necessary to either co-express an ion channel, such as a potassium inward rectifier, that
5 can maintain the resting transmembrane potential of the cell to around -90 mV, or identify a cell
line that endogenously expresses similar ion channels. Cell types of this type include RBL cells
andHEK-293 cells.
In other cases it may be necessary to use the expression of a second ion channel, in
conjunction with electrical stimulation to drive the cell membrane to a specific transmembrane
10 potential, to enable the first ion channel of interest to be assayed. Examples of this situation occur
when assaying non-voltage regulated ion channels such as ligand-gated channels. Co-expression of
a voltage regulated sodium channel, for example in conjunction with electrical stimulation can be
used to set the transmembrane potential to transmembrane potentials of between about +10 to +60
mV. By comparison, co-expression of voltage regulated potassium channels in conjunction with
15 electrical stimulation can set the transmembrane potential to transmembrane potentials of between
about —90 to -30 mV. These approaches thus enable the effective manipulation of the
transmembrane potential over a relatively wide range thereby enabling the analysis of virtually any
ion channel.
Typically when using this co-expression approach it is necessary to re-screen any hits
20 obtained with the cell line co-expressing both ion channels, with the cell line expressing only the
ion channel used to set the transmembrane potential. This enables drugs that affect this second ion
channel to be differentiated from those that actually influence the ion channel of interest.
Alternatively selective toxins such as TTX can be used to selectively inhibit one class of ion
channel.
25 b) Transfection of ion channels
Nucleic acids used to transfect cells with sequences coding for expression of the ion
channel of interest are typically in the form of an expression vector including expression control
sequences operatively linked to a nucleotide sequence coding for expression of the channel. As
used, the term nucleotide sequence coding for expression of a channel refers to a sequence that,
30 upon transcription and translation of mRNA, produces the channel. This can include sequences
containing, e.g., introns. As used herein, the term expression control sequences refers to nucleic
acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively
linked. Expression control sequences are operatively linked to a nucleic acid sequence when the
expression control sequences control and regulate the transcription and, as appropriate, translation
35 of the nucleic acid sequence. Thus, expression control sequences can include appropriate
promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-
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WO 02/08748 PCT/US01/21652
encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene
to permit proper translation of the mRNA, and stop codons.
Methods which are well known to those skilled in the art can be used to construct expression
vectors containing the ion channel coding sequence, operatively coupled to appropriate localization
5 or targeting domains and appropriate transcriptional / translational control signals. For example by
reference to the sequence accession numbers, or references in Tables 1 to 3, one or ordinary skill in
the art can identify the sequence of the ion channel of interest. These methods include in vitro
recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic
recombination. (See, for example, the techniques described in Maniatis, et ah y Molecular Cloning
10 A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989). Many commercially available
expression vectors are available from a variety of sources including Clontech (Palo Alto, CA),
Stratagene (San Diego, CA) and Invitrogen (San Diego, CA) as well as and many other
commercial sources.
A contemplated version of the method is to use inducible controlling nucleotide sequences
15 to produce a sudden increase in the expression of the ion channel of interest e.g., by inducing
expression of the channel. Example inducible systems include the tetracycline inducible system
first described by Bujard and colleagues (Gossen and Bujard (1992) Proc. Natl. Acad. Sci USA 89
5547-5551, Gossen et al. (1995) Science 268 1766-1769) and described in U.S. Patent No
5,464,758.
20 Transformation of a host cell with recombinant DNA may be carried out by conventional
techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E.
colU competent cells that are capable of DNA uptake can be prepared from cells harvested after
exponential growth phase and subsequently treated by the CaCl2 method by procedures well
known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be
25 performed after forming a protoplast of the host cell or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate
co-precipitates, conventional mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can
also be co-transfected with DNA sequences encoding the ion channel, and a second foreign DNA
30 molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic cells and express the ion channel.
{Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, a
eukaryotic host is utilized as the host cell as described herein.
35 Selection of stable clones will typically be made on the basis of successful expression of
the ion channel of interest at sufficient level to enable it's facile detection. In many cases this
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WO 02/08748 PCT/US01/21652
analysis will require functional characterization of individual clones to identify those that exhibit
appropriate electrophysiological characteristics consistent with expression of the clone of interest.
This analysis can be completed via the use of patch clamping, or via the measurement of
transmembrane potentials using transmembrane potential sensitive dyes as described below. An
5 advantage to the use of this latter method is that it is compatible with fluorescence activated cell
sorting and provides for the rapid analysis of many thousands of individual clones per second. In
some cases where the sodium channel is electrically silent in the resting cell, confirmation of
expression can also be readily achieved by immunochemistry using antibodies raised against the
native ion channel, or a defined epitope introduced in the ion channel via molecular techniques as
1 0 described above.
In cases where cells are transfected with a first ion channel of interest, and a second ion
channel to set the transmembrane potential, optimization of the relative expression of both ion
channels is important. Typically the optimal relative expression of the two ion channels is
determined empirically by selecting clones that provide the maximum dynamic range and minimal
1 5 statistical variation in their response.
VII . Measurement of Transmembrane potentials
Transmembrane potential changes and the measurement of specific ion channels
conductance via the use of the present invention can be detected by use of any of the known means
of measuring transmembrane potential or ion movement. These methods include, for example,
20 patch clamping (Hamill et al, Pfluegers Arch. 391:85-100, 1981), FRET based voltage sensors,
electrochromic transmembrane potential dyes (Cohen et al., Annual Reviews of Neuroscience 1:
171-82, 1978), transmembrane potential redistribution dyes (Freedman and Laris, Spectroscopic
membrane probes Ch 16, 1988), extracellular electrodes (Thomas et al., Exp. Cell Res. 74: 61-66,
1972), field effect transistors (Fromherz et al., Science 252: 1290-1293, 1991) , radioactive flux
25 assays, ion sensitive fluorescent or luminescent dyes, ion sensitive fluorescent or luminescent
proteins, the expression of endogenous proteins or the use of reporter genes or molecules.
Preferred methods of analysis for high throughput screening typically involve the use of
optical readouts of transmembrane potential, or ion channel conductance. Such methods include
the use of transmembrane potential or ion sensitive dyes, or molecules, that typically exhibit a
30 change in their fluorescent or luminescent characteristics as a result of changes in ion channel
conductance or transmembrane potential.
A preferred optical method of analysis for use with the present invention has been
described in U.S. patent No 5,661, 035 issued August 26, 1997). This approach typically comprises
two reagents that undergo energy transfer to provide a ratiometric fluorescent readout that is
35 dependent upon the transmembrane potential. Typically the approach uses a voltage sensing
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WO 02/08748 PCT/US01/21652
lipophilic dye and a voltage insensitive fluorophore associated with a cell membrane, (see
Gonzalez et al. Drug Discovery Today 4:43 1-439, 1999).
In one embodiment, two dye molecules, a coumarin-linked phospholipid (CC2-DMPE) and
an oxonol dye such as bis-(l,2-dibutylbarbituric acid) trimethine oxonol [DiSBAC4(3)], are loaded
5 into the plasma membrane of cells. CC2-DMPE partitions into the outer leaflet of the plasma
membrane where it acts as a fixed FRET donor to the mobile, voltage sensitive oxonol acceptor.
Cells with relatively negative potentials inside will push the negatively charged oxonol to the outer
leaflet of the plasma membrane, resulting in efficient FRET (i.e. quenching of the coumarin donor
and excitation of the oxonol acceptor). Depolarization results in rapid translocation of the oxonol
10 to the inner surface of the plasma membrane, decreasing FRET. Because FRET can only occur
over distances of less than 100 A, excitation of the coumarin results in specific monitoring of
oxonol movements within the plasma membrane.
The response times for these assays is readily altered by increasing or decreasing the
hydrophobicity of the oxonol. For example, the more hydrophobic dibutyl oxonol DiSBAC4(3)
15 has a time constant of approximately 10 ms, significantly faster than the less hydrophobic diethyl
oxonol DiSBAC2(3).
Loading of the dyes is typically achieved at room temperature prior to the start of
transmembrane potential measurements. Typically cells are loaded sequentially with the coumarin
lipid followed by the oxonol. Typical loading concentrations for coumarin lipids range from about
20 4 to 15 |LiM (final concentration) and staining solutions are typically prepared in Hanks Balanced
salt solution with 10 mM HEPES, 2g/L glucose and about 0.02% Pluronic-127 at a pH of around
7.2 to 7.4. Loading is usually acceptable after about 30 minutes incubation, after which excess dye
may be removed if desired. Oxonol dyes are typically loaded at a concentration between 2 and 10
jlxM for 25 minutes at room temperature, the more hydrophobic DiSBAC4(3) is usually loaded in
25 the presence of 2-3 pM Pluronic-127. Optimal loading concentrations vary between cell types and
can be empirically determined by routine experimentation. Typically such optimization
experiments are conducted by systematically titrating the concentrations of the first reagent, and
then for each concentration tested, titrating the concentration of the second reagent. In this way it is
possible to obtain both the optimal loading concentrations for each reagent, and the optimal
30 relative ratio to achieve a maximal signal to noise ratio.
In some cases it may be preferred to add, or load one, or more of the FRET reagents with
one or more light absorbing substances in order to reduce undesired light emission, as for example
described in commonly owned U.S. Patent Application No. 09/118,497, filed July 17, 1998; U.S.
Patent Application No. 09/120,516, filed July 21, 1998, and U.S. Patent Application No.
35 09/122,477 filed July 23, 1998.
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WO 02/08748 PCT/US01/21652
FRET based voltage sensors may also be derived from the use of other membrane targeted
fluorophores in conjunction with a mobile hydrophobic donor or acceptor. Other such
compositions are disclosed, for example, in U.S. Patent Application No. 09/459,956, filed
December 13, 1999.
5 Suitable instrumentation for measuring transmembrane potential changes via optical
methods includes microscopes, multiwell plate readers and other instrumentation that is capable of
rapid, sensitive ratiometric fluorescence detection. A preferred instrument of this type is described
in U.S. Patent application 09/118,728 filed July 17, 1998. This instrument (the Voltage/Ion Probe
Reader or VIPR™) is an integrated liquid handler and kinetic fluorescence reader for 96-well and
10 greater multiwell plates. The VIPR™ reader integrates an eight channel liquid handler, a multiwell
positioning stage and a fiber-optic illumination and detection system. The system is designed to
measure fluorescence from a column of eight wells simultaneously before, during and after the
introduction of liquid sample obtained from another microtiter plate or trough. The VIPR™ reader
excites and detects emission signals from the bottom of a multiwell plate by employing eight
15 trifurcated optical bundles (one bundle for each well). One leg of the trifurcated fiber is used as an
excitation source, the other two legs of the trifurcated fiber being used to detect fluorescence
emission. A ball lens on the end of the fiber increases the efficiency of light excitation and
collection. The bifurcated emission fibers allow the reader to detect two emission signals
simultaneously and are compatible with rapid signals generated by the FRET-based voltage dyes.
20 Photomultiplier tubes then detect emission fluorescence, enabling sub-second emission ratio
detection.
VIII. Stimulation Protocols
In one aspect, the present invention includes methods for modulating the transmembrane
potentials of living cells via electrical stimulation, and the use of these methods for assaying the
25 activity of virtually any ion channel or transporter system.
a) Measurement of specific channel conductances
L Assay of sodium channels
A variety of different isoforms of mammalian voltage dependent sodium channels have
been identified, and are summarized below in Table 1. These channels can be classified into three
30 main groups (for review see Goldin, Annals N.Y. Academy of Sciences 868:38-50, 1999).
35
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PCT/US01/21652
Table 1
Sodium Channel Sub-type Summary
Channel Name &
Sub-type / Alternate names
Tissue Distribution
Accession
Gene Symbol
Number
SCN1A (Navl.l)
Rat I (rat)
CNS / PNS
X03638
HBSCI (human)
CNS
X65362
GPB1 (Guinea pig)
CNS
AF003372
SCN2A (Navl.2)
■■■•i: ' : .• '
Rat II (rat)
CNS
X03639
HBSCII (human)
CNS
X65361
HBA (human)
CNS
M94055
Nav 1.2A
Rat II A
CNS
X61149
SCN3A (Nav 1.3)
Rat III (rat)
CNS
Y00766
SCN4A (Navl.4)
SkMl 5 (lxI (rat)
skeletal muscle
M26643
SkMl ^human^
<J JVwlt Id J. lilUOviv
M81758
SCN5A (Navl.5)
QVA/T9 ^roti
„
skeletal muscle /
M27902
heart
xj. i ^xiumdii ^
heart
A/1779
1V1 / / Z, J *J
SCN8A rflVavl 6\
NaCh6 (rat)
CNS / PNS
L39018
PN4a f rat"!
CNS / PNS
AF049239A
Scn8a (mouse)
CNS
U26707
Scn8a (human)
CNS
AF050736
Cerlll (Guinea pig)
CNS
AF003373
SCN9A (Navl/7) ^
PN1 (rat)
PNS
U79568
HNE-Na (human)
thyroid
X82835
Nas (rabbit)
Schwann cells
U35238
SCN10A NavL8
Pi-
SNS (rat)
PNS
X92184
PN3 (rat)
PNS
U53833
SNS (mouse)
PNS
Y09108
8CN6ANav2.1
Na2.1 (human)
Heart, uterus muscle
M91556
SCN7A Nav2.2
Na-G (rat)
astrocytes
M96578
SCLll(rat)
PNS
Y09164
Nav2.3
Na2.3 (mouse)
Heart, uterus muscle
L36179
Nav3.1
NaN (rat)
PNS
AF059030
SCNlBNapi.l
P-l (rat)
CNS
M91808
P-l (human)
CNS
L10338
SCN2BNap2.1
p-2 (rat)
CNS
U37026
p-2 (human)
CNS
AF007783
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The voltage-dependent sodium channels in Table 1 vary widely in their voltage
dependency and inactivation and activation kinetics. Voltage-gated sodium channels have many
different conformations, which can be classified into three states. (1) The resting state, in which
the channel is closed and no current can flow. This is the typical state when a sodium channel is
5 expressed in a cell with a resting transmembrane potential of below about -60 mV. The channel
can be rapidly driven into the open state by depolarization, usually to a transmembrane potential of
above about —50 mV. (2) The activated state, in which the channel is open and ions can pass
through. Because the intracellular concentration of sodium is low in a normal resting cell, while
the extracellular concentration is high, sodium ions flow into the cell and drive the transmembrane
10 potential more positive. The open state has a short lifetime, generally on the order of one
millisecond, after which it passes into the inactivated state. (3) The inactivated state, in which a
channel has closed and ions can not pass through the channel. The channel cannot be directly
opened once in the inactivated state. It will first go to the resting state, which occurs if the
transmembrane potential is held very negative (generally below -80 mV) for several milliseconds.
15 The time constants and threshold potentials for transitions between these three states vary greatly
between channel subtypes.
During these experiments, the response will be compared for cells with active channels,
and for cells in which the channels are pharmacologically blocked. If a suitable pharmacological
agent is not available, the blocked state can be emulated with an un-transfected cell line. The
20 optimal stimulus parameters will yield the smallest coefficient of variation of the difference in
signals of the two cell populations.
i) Assays for voltage-dependent sodium channels in an inactivated state
Preferred cells include those with resting transmembrane potentials above the activation
threshold for the ion channel of interest, and in which there are no other ion channels expressed.
25 Cells meeting these criteria include CHL and LTK(-) cells. After choosing a target ion channel,
cells are transfected and clones are selected as described in section III. Alternatively, a cell line
that endogenously expresses the channel of interest, and low levels of other channels, could be
used. For example, the CHO-K1 cell line expresses a voltage-gated sodium channel, and very low
levels of other ion channels. Cells are plated into multiwell microtiter plates, cultured, and stained
30 with voltage-sensitive dyes as described in section IV prior to initiating electrical stimulation.
Initial experiments are typically carried out in a 96-well multiwell plate, with an equal number of
cells in each well. Generally columns of eight wells are simultaneously stimulated under identical
conditions to provide statistically significant data on the variation in cellular response.
An optimal electrical stimulation protocol should hyperpolarize part of the plasma
35 membrane of the majority of the cells long enough to release the sodium channels from
inactivation, prior to providing an activating depolarization, without electroporating or killing the
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cells. Typically this requires sustained transmembrane potentials of around -60 to -80 mV for
periods ranging from about 0.5 to about 20 ms to be created within the cell.
A preferred stimulation protocol that achieves this effect is biphasic, so that ion channels
present on both the extreme edges of the cells are released from inactivation as the biphasic
5 waveform reverses polarity. Typically one would start out with initial conditions using a biphasic
square wave kernel of 5 msec per phase and an amplitude of 25 V/cm. The kernel would be
repeated at a regular rate of about 20 Hz for a total train duration of about three seconds. One
would then optimize the pulse amplitude (up to a maximum of about 60 V/cm), duration (in the
range of 0.1 to 50 ms), and then frequency (in the range of 0 to 1kHz). If necessary changes in the
10 pulse shape could also be explored to determine if these resulted in more efficient electrical
stimulation. The optimal stimulus parameters will yield the maximum cellular stimulation
(compared to cells with the channel blocked, or not present) with smallest coefficient of variation
of the signal among the different test wells, at the lowest electric field strength, and at the lowest
duty cycle for passage of current through the electrodes. After a particular set of parameters is
15 chosen, a titration of staining concentrations for the voltage sensor dye(s) should be performed as
described above, to further optimize the signal size and coefficient of variation of the responses.
These procedures (dye concentrations, electric field strength, and stimulus duration and frequency)
can be iterated to further optimize the signal.
ii) Assays for sodium channels normally in the resting state
20 Preferred cells include those with resting transmembrane potentials below the activation
threshold for the ion channel of interest, and in which the expression of other ion channels is
largely confined to a few characterized ion channel types. Cells of this type include HEK-293 and
RBL cells as well as Fll and HL5 cells. After choosing a target ion channel, cells are transfected
with the ion channel of interest and clones are selected as described above. Alternatively, as in the
25 case of Fll and HL5 cells, endogenous sodium channels can be used. After selection and
characterization, cell clones are plated into multiwell microtiter plates and stained with voltage-
sensitive dyes as described above. As previously, initial experiments are typically carried out in a
96-well multiwell plate, with an equal number of cells in each well. Generally columns of eight
wells are simultaneously stimulated under identical conditions to provide statistically significant
30 data on the variation in cellular response.
A number of assay approaches are possible depending on the expression level of the
sodium channel of interest in the cell. For high levels of voltage-dependent sodium channel
expression, the sodium current can be large enough to create a large transmembrane potential
change after a single channel activation/inactivation sequence. In these cases small positive
35 perturbations in the transmembrane potential created via electrical stimulation can be sufficient to
activate enough sodium channels that the subsequent sodium ion entry depolarizes the entire cell
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thereby activating all the sodium channels. The stimulus field should typically be applied for a
time long enough to activate the channels, but not so long as to interfere with the subsequent ion
flux. After the cell transmembrane potential has re-polarized, the stimulation procedure can be
repeated. Subsequent stimulation events can be identical to the first, or varied to examine time-
5 dependent properties of the channels.
Typically one would start out with initial conditions using a biphasic square wave kernel of
500 jus per phase and an amplitude of 10 V/cm. One would then optimize the pulse amplitude
(between 5 and 60 V/cm) and duration (between 0.1 and 1 ms). If necessary changes in the pulse
shape could also be explored to determine if these resulted in more efficient electrical stimulation.
10 The optimal stimulus parameters will yield the maximum cellular stimulation with smallest
coefficient of variation of the signal among the different test wells, at the lowest electric field
strength, and at the lowest duty cycle for passage of current through the electrodes. After a
particular set of parameters is chosen, a titration of staining concentrations for the voltage sensor
dye(s) should be performed as described above, to further optimize the signal size and coefficient
15 of variation of the responses. These procedures (dye concentrations, electric field strength, and
stimulus duration and frequency) can be iterated to further optimize the signal.
Often it will be necessary to use cells whose expression of sodium channels is too low to
give acceptable signal sizes from single stimuli. It may also be desirable to maintain a large signal
over an extended period of time. In these cases, the cells can be given pulse trains as described for
20 channels held above the activation potential. With biphasic stimulus pulses, the sodium channels
can be activated independent of the starting transmembrane potential. By keeping the inter-pulse
interval shorter than the membrane time constant, each stimulus will drive current into the cell
until an equilibrium between inward and outward currents is established. This voltage deviation
will be maintained as long as the stimulus train continues.
25 The stimulation protocols in this case are essentially the same as described for cells whose
resting potential is above the inactivation threshold. In general, a series of initial experiments are
conducted using a biphasic square wave kernel repeated at a regular rate for a fixed train duration.
The pulse duration varies from about 1 \is to about 1 s, and more preferably from about 1 00 \xs to
about 20 ms. The pulse amplitude varies from 0 V/cm to about 60 V/cm, and more preferably
30 from 10 V/cm to 50 V/cm. The frequency of stimulation varies between 0 Hz (i.e. a single pulse)
and 1 00 kHz, and more preferably from 0 Hz to about 1 kHz. The pulse train varies between 0 s
(i.e. a single pulse) and about 100 s, and more preferably between 0 s and 10 s. The optimal
stimulus parameters will yield the maximum transmembrane potential changes (compared to cells
with the channel blocked, or not present) and smallest coefficient of variation of the signal among
35 the test wells, at the lowest electric field strength. After a particular set of parameters is chosen, a
titration of staining concentrations for the voltage sensor dye(s) is typically performed as described
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PCT/US01/21652
above to further optimize the signal size and coefficient of variation of the responses. These
procedures (dye concentrations, electric field strength, and stimulus duration and frequency) can be
iterated to further optimize the signal.
b) Potassium channels
5 Voltage-dependent potassium channels repolarize nerve and muscle cells after action
potential depolarization. They also play important regulatory roles in neural, muscular, secretory,
and excretory systems. Most cells actively maintain a high intracellular potassium concentration,
so that the reversal transmembrane potential for potassium is around -90 mV. Potassium typically
flows out of the cell, so that opening more potassium-selective channels tends to drive the
10 transmembrane potential more negative, in contrast to sodium channel opening that typically drives
the transmembrane potential more positive.
A summary of the numerous potassium sub-types is presented in Table 2 below.
Table 2
Potassium Channel Sub-type Summary
Channel Type
Sub-type / Alternate names
Accession
Number
Reference
ATP regulated
• ; V
rKirl.l (ROMK1) (rat)
U12541
U.S. Patent 5,356,775
hKirl . 1 (ROMK1 )(human)
U.S. Patent 5,882,873
Kirl.2
U73191
Kirl.3
U73193
I.
B-cell
U.S. Patent 5,744,594
II.
h3IR
U.S. Patent 5,917,027
III.
HuKATP-1
EP 0 768 379 Al
Constitiiiively
Active . • ..
Kir2.1(IRKl)
U12507
U.S. Patent 5,492,825
U.S. Patent 5,670,335
Kir2.2
X78461
Kir2.3
U07364
G-proteiri
Regulated
Kir3.1 (GIK1, KG A)
U01071
U.S. Patent 5,728,535
Kir3.2
U11859
U.S. Patent 5,734,021
Kir3.3
U11869
U.S. Patent 5,744,324
Kir3.4 (CIR)
X83584
U.S. Patent 5,747,278
Kir4.1(BIR10)
X83585
Kir5.1(BIR9)
X83581
Kir6.1
D42145
Kir6.2
D5081
Kir7.1
EP 0 922 763 Al
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WO 02/08748
PCT/US01/21652
Table 2
Potassium Channel Sub-type Summary
Channel Type
Sub-type / Alternate names
Accession
Number
Reference
Voltage Regulated
KCNA1
hKvl.l (RCK1, RBK1, MBK1,
MK1, HuKl)
LO2750
KCNA2
Kvl.2 (RBK2, RBK5, NGK1,
KCNA3
Kvl.3 (KV3, RGK5, HuKffl,
KCNA4
Kvl.4 (RCK4, RHK1, HuKTT)
TVOTVT A C
TV- , 1 C / P T/'\n T T T 1 TTT/'1\
Kvl.5 (KV1, HPCN1, HK2)
KLNAo
TV-. , 1 *r /'T/'t TJ f~~~<T/~ O T TT> TV O "\
Kvl.o (KV2, RCK2, HBK2)
TV p\T a T
TV.., 1 T /A /f T/" T>TVZT TT«T/ r ^\
Kv 1.7 (Mko, RK6, HaKo)
U.S. Patent 5,559,009
Jiv2 {iSftab)
KCJN131
TV O 1 /T~\T» TT' 1 oi_ 1_\
Kv2.1(DRKl, mShab)
M64228
TV OT^TD O
TV, ,0 O //^TATl TV 1 \
Kv2.2 (CDRK1)
K channel 2
U.S. Patent 5,710,019
AvJ (Shaw)
KCNC1
Kv3.1 (NGK2)
KCJNC2
Kv3.2 (RKShlllA)
KCNC3
Kv3.3 (KSliIIID)
X60796
KCNC4
Kv3.4 (Raw3)
Jiv¥ (Shal)
KCND1
Kv4.1 (mShal, KShlVA)
M64226
KCND2
Kv4.2 (RK5, Rat Shal l)
KCND3
TV . , A "> /TV 0 1~ YT m ~\
Kv4.3 (KSnlVB)
l^TV »C 1 /TTVO\
nKv5.1(IK8)
WO 99/41372
Kvo.l (Kl3)
TV, T ' r 7
Kv7
Kv8.l
Kv9
Delayed Rectifier
KvLQTl
AF000571
U.S. Patent 5,599,673
HERG (erg)
U04270
PCT WO99/20760
Calcium regulated
Ca^ Regulated
Big
BKCa (hSLO)
UH717
HBKb3 (p-subunit)
PCT W099/42575
Maxi-K
U.S. Patent 5,776,734
U.S. Patent 5,637,470 |
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WO 02/08748
PCT/US01/21652
Table 2
Potassium Channel Sub-type Summary
Channel Type
Sub-type / Alternate names
Accession
Number
Reference
c<2~ Kegulatect-
small
KCNN1
SKCal
U69883
KCNN2
SKCa2
U69882
KCNN3
SKCa3
U69884
KCNN4
SKCa4 (IKCal)
Muscle Nerve 1999
22(6) 742-50
TWIK1
U33632
Potassium channels show enormous diversity in terms of activation and inactivation time
constants and voltage dependencies. In general, voltage-dependent potassium channels show voltage
dependence similar to sodium channels, being closed at very negative potentials and opening above a
5 certain threshold. Potassium channels may have multiple resting states, multiple inactivated states,
and typically a single activated state. Unlike voltage-dependent sodium channels, transitions are
allowed between most states. These transitions are activation (moving from a resting to the open
state), deactivation (moving from the open state to a resting state), inactivation (moving from a resting
or open state to an inactivated state), release from inactivation (moving from an inactivated state to a
10 resting state), and flickering (moving from an inactivated state to the open state). There is a great
diversity in the thresholds of the transitions, and in the voltage dependencies of the transition rates.
Activation time constants range from 0.1 to 1000 ms with threshold activation potentials from -80 to
+20 mV. Inactivation time constants range from 0.1 to infinity (i.e. no inactivation) with threshold
potentials from -60 to 0 mV. Time constants for release from inactivation range from 0.5 ms to 100
15 ms with threshold potentials from -70 to 0 mV.
Stimulus protocols necessary to obtain measurable channel- dependent signals are
somewhat dependent upon the specific properties of the channel in question. Because of the
diversity in parameters in voltage-dependent potassium channels, the optimization of an electrical
stimulation protocol may take several iterations.
20 During these experiments, the response will be compared for cells with active channels,
and for cells in which the channels are pharmacologically blocked. If a suitable pharmacological
agent is not available, the blocked state can be emulated with an un-transfected cell line. The
optimal stimulus parameters will yield the smallest coefficient of variation of the difference in
signals of the two cell populations.
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WO 02/08748 PCT/US01/21652
Assays using direct stimulation of the potassium channel
Voltage Regulated potassium Channels
Because potassium channels generate outward currents, activating the channels causes
negative transmembrane potential changes. Under physiological conditions, the reversal potential
5 for potassium is around -90 mV. Because cells expressing only a voltage-dependent potassium
channel generally have resting potentials near the activation threshold, direct stimulation should
work for those voltage-dependent potassium channels which have activation thresholds above
about -50 mV. While small negative deflections in the transmembrane potential (less than 40 mV
change) can be reliably detected using the FRET voltage-sensitive dyes, it is often preferable to
10 perform high-throughput screens with larger signals.
Preferred cell types include those cells that express a minimal level of other ion channels,
such as CHO-K1, CHL, and LTK(-). The transfection and selection of clones expressing ion
channels of interest will generally be performed as described above for sodium ion channels
normally in the resting state. Alternatively, a cell line which endogenously expresses the channel
15 of interest could be used. The labeling and measurement of cells with transmembrane potential
dyes will generally be performed as described for sodium ion channels normally in the resting
state.
The stimulation protocol will advantageously depolarize part of the plasma membrane long
enough to activate the voltage-dependent potassium channels. Unlike the case for voltage-
20 dependent sodium channels, voltage-dependent potassium channels will typically pass current
during the depolarizing phase of the stimulus pulse. On the side of the cell where the
transmembrane potential is driven in a negative direction, the potassium channels release from
inactivation (if the channel in question experiences voltage-dependent inactivation). On the side of
the cell where the transmembrane potential is driven in a positive direction, potassium channels
25 activate and pass outward current. Thus, the stimulus pulse duration should not greatly exceed the
inactivation time. The potassium current tends to drive the average transmembrane potential
negative of the resting potential. After the stimulus pulse, the transmembrane potential will
exponentially relax to the resting potential. By repeating the stimulus after a time shorter than the
membrane time constant, the average cell membrane can be driven further negative. Using a train
30 of stimuli, a large and sustained signal can be obtained.
A preferred stimulation protocol that achieves this effect is biphasic, so that ion channels
present on both the extreme edges of the cells can participate in enabling potassium ion movement.
Typically one would start out with initial conditions using a biphasic square wave kernel of 5 msec
per phase and an amplitude of 25 V/cm. The kernel would be repeated at a regular rate of about 20
35 Hz for a total train duration of about three seconds. One would then optimize the pulse amplitude,
duration, and then frequency. If necessary changes in the pulse shape could also be explored to
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WO 02/08748 PCT/US01/21652
determine if these resulted in more efficient electrical stimulation. The optimal stimulus
parameters will yield the maximum average transmembrane potential change (compared to cells
with the channel blocked, or not present) with smallest coefficient of variation of the signal among
the different test wells, at the lowest electric field strength, and at the lowest duty cycle for passage
5 of current through the electrodes. After a particular set of parameters is chosen, a titration of
staining concentrations for the voltage sensor dye(s) should be performed as described above, to
further optimize the signal size and coefficient of variation of the responses. These procedures
(dye concentrations, electric field strength, and stimulus duration and frequency) can be iterated to
further optimize the signal.
10 2) Inward-rectifier potassium channels
Contrary to its name, the function of the inward rectifier channel is not to allow potassium
into the cell. Inward flow of potassium can only occur (1) when the transmembrane potential falls
below the potassium equilibrium potentials, or (2) if the extracellular potassium concentration
rises. Neither situation normally occurs, because (1) under normal physiological conditions, since
15 potassium is the ion with the most negative reversal potential, no ionic current can drive the
potential more negative than the potassium reversal potential, and (2) except under pathological
conditions, the extracellular potassium concentration is tightly controlled. However, using
electrical stimulation, parts of the cell membrane can be driven below Vk ? promoting potassium
ion entry into the cell. This will cause a net positive transmembrane potential change and can be
20 detected as a positive signal. To develop and optimize an assay for blockers of the inward rectifier,
one could therefore follow the following procedure.
Preferred cell types include those cells that express a minimal level of other ion channels,
such as CHO-K1, CHL, and LTK(-). The transfection and selection of clones expressing ion
channels of interest will generally be performed as described above for sodium ion channels
25 normally in the resting state. Alternatively, a cell line which endogenously expresses the channel
of interest could be used. The labeling and measurement of cells with transmembrane potential
dyes will generally be performed as described for sodium ion channels normally in the resting
state.
A preferred stimulation protocol uses a biphasic kernel, so that ion channels present on
30 both the extreme edges of the cells participate. Typically one would start out with initial conditions
using a biphasic square wave kernel of 5 msec per phase and an amplitude of 25 V/cm. The kernel
would be repeated at a regular rate of about 20 Hz for a total train duration of about three seconds.
One would then optimize the pulse amplitude, duration, and then frequency. If necessary changes
in the pulse shape could also be explored to determine if these resulted in more efficient electrical
35 stimulation. The optimal stimulus parameters will yield the maximum cellular stimulation
(compared to cells with the channel blocked, or not present) with the smallest coefficient of
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WO 02/08748 PCT/US01/21652
variation of the signal among the different test wells, at the lowest electric field strength, and at the
lowest duty cycle for passage of current through the electrodes. After a particular set of parameters
is chosen, a titration of staining concentrations for the voltage sensor dye(s) should be performed
as described above, to further optimize the signal size and coefficient of variation of the responses.
5 These procedures (dye concentrations, electric field strength, and stimulus duration and frequency)
can be iterated to further optimize the signal.
iii) Assays using a voltage-dependent sodium counter-channel
This method involves the use of a cell line expressing the voltage-dependent potassium
channel of interest and which also expresses a voltage-dependent sodium channel. In this method
1 0 the approach is to use electrical stimulation protocols designed to specifically activate the voltage
dependent sodium channel. In this case electrical stimulation causes sodium ions to enter the cell,
causing a positive voltage change. The presence of the potassium channel of interest will tend to
suppress the positive response of the sodium channel by allowing potassium ions to leave the cell.
The assay takes advantage of the absence of outward current when a test chemical blocks the
15 potassium channel, thereby restoring the large positive voltage response normally induced by
activation of the sodium channels. The optimization of the balance of currents is important in this
method to ensure that the assay is sensitive to potassium channel blockade. If the sodium current is
too small relative to the potassium current, the dose-response curve for the potassium channel
blocker will be shifted towards higher concentrations. For example, in the extreme case where the
20 potassium current is 100 times larger than the sodium current, 99% of the potassium channels
would have to be blocked in order to get a 50% response from the sodium channels.
Because this method involves driving a voltage-dependent sodium channel with repetitive
pulses, the protocol development is essentially the same as described above for voltage-activated
sodium channels in an inactivated state. Typically one would start out with initial conditions using
, 25 a biphasic square wave kernel of 5 msec per phase and an amplitude of 25 V/cm. The kernel
would be repeated at a regular rate of about 20 Hz for a total train duration of about three seconds.
One would then optimize the pulse amplitude, duration, and then frequency. If necessary changes
in the pulse shape could also be explored to determine if these resulted in more efficient electrical
stimulation. The optimal stimulus parameters will yield the maximum cellular stimulation
30 (compared to cells with the channel blocked, or not present) with smallest coefficient of variation
of the signal among the different test wells, at the lowest electric field strength, and at the lowest
duty cycle for passage of current through the electrodes. After a particular set of parameters is
chosen, a titration of staining concentrations for the voltage sensor dye(s) should be performed as
described above, to further optimize the signal size and coefficient of variation of the responses.
35 These procedures (dye concentrations, electric field strength, and stimulus duration and frequency)
can be iterated to further optimize the signal.
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WO 02/08748 PCT/US01/21652
In this assay format, there will ideally be no (or a very small) response to stimulation in the
absence of channel block, because the potassium current will counteract the sodium current.
Therefore, to optimize the stimulus conditions, it will be necessary to compare responses with and
without the activity of the potassium channel. Ideally, this will be accomplished using a selective
5 blocker of the potassium channel. In those cases where such a blocker is yet unknown, it will be
possible to use the cell line containing only the sodium counter-channel.
Because this assay format involves two ion channels, modulators of either channel will
affect the voltage response. In this case, a hit (a blocker of the potassium channel) will restore the
voltage response. The screening format automatically ignores compounds which block only the
10 sodium channel. However, stimulation of the cells in the presence of compounds which block both
channels will also result in no voltage deflection, suggesting that the compound is inactive.
Because compounds of this type may be of interest, a method to unmask them is also available. By
performing the identical compound screen using the parent cell line, which contains the sodium
channel but not the potassium channel, blockers of the sodium channel can be found. Compounds
15 which are found to block the sodium channel can then be tested separately to find if they have
activity against the potassium channel.
c) Assay of calcium channels
Calcium channels are generally found in many cells where, among other functions, they
play important roles in signal transduction. In excitable cells, intracellular calcium supplies a
20 maintained inward current for long depolarizing responses and serves as the link between
depolarization and other intracellular signal transduction mechanisms. Like voltage-gated sodium
channels, voltage-gated calcium channels have multiple resting, activated, and inactivated states.
Multiple types of calcium channels have been identified in mammalian cells from various
tissues, including skeletal muscle, cardiac muscle, lung, smooth muscle and brain, [see, e.g., Bean,
25 B. P. (1989) Ann. Rev. Physiol. 51:367-384 and Hess, P. (1990) Ann. Rev. Neurosci. 56:337]. The
different types of calcium channels have been broadly categorized into four classes, L-, T-, N-, and
P-type, distinguished by current kinetics, holding potential sensitivity and sensitivity to calcium
channel agonists and antagonists. Four subtypes of neuronal voltage-dependent calcium channels
have been proposed (Swandulla, D. et al., Trends in Neuroscience 14:46, 1991).
30 The cDNA and corresponding amino acid sequences of the al, a2, P and y subunits of the
rabbit skeletal muscle calcium channel have been determined [see, Tanabe et al. (1987) Nature
328:313-318; Ruth et al. (1989) Science 245:1115-1118; and U.S. Pat. No. 5386,025]. In addition,
the cDNA and corresponding amino acid sequences of al subunits of rabbit cardiac muscle
[Mikami, A. et al. (1989) Nature 340:230-233] and lung [Biel, M. (1990) FEBS Letters 269:409-
35 412] calcium channels have been determined. In addition, cDNA clones encoding a rabbit brain
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WO 02/08748 PCT/US01/21652
calcium channel (designated the BI channel) have been isolated [Mori, Y. et al. (1991) Nature
350:398-4021,
Partial cDNA clones encoding portions of several different subtypes, referred to as rat
brain class A, B, C and D, of the calcium channel al subunit have been isolated from rat brain
5 cDNA libraries [Snutch, T. et al. (1990) Proc. Natl. Acad. Sci. USA 87:3391-3395]. More recently
full-length rat brain class A [Starr, T. et al. (1991) Proc. Natl. Acad. Sci. USA 88:5621-5625] and
class C [Snutch, T. et al. (1991) Neuron 7:45-57] cDNA clones have been isolated. Although the
amino acid sequence encoded by the rat brain class C DNA is approximately 95% identical to that
encoded by the rabbit cardiac muscle calcium channel al subunit-encoding DNA, the amino acid
10 sequence encoded by the rat brain class A DNA shares only 33% sequence identity with the amino
acid sequence encoded by the rabbit skeletal or cardiac muscle al subunit-encoding DNA. A
cDNA clone encoding another rat brain calcium channel al subunit has also been obtained [Hui,
A. et al. (1991) Neuron 7:35-44]. The amino acid sequence encoded by this clone is approximately
70% homologous to the proteins encoded by the rabbit skeletal and cardiac muscle calcium
15 channel DNA. A cDNA clone closely related to the rat brain class C al subunit-encoding cDNA
and sequences of partial cDNA clones closely related to other partial cDNA clones encoding
apparently different calcium channel al subunits have also been isolated [see Snutch, T. et al.
(1991) Neuron 7:45-57; Perez-Reyes, E. et al. (1990) J. Biol. Chem. 265:20430; and Hui, A. et al.
(1991) Neuron 7:35-44].
20 For known calcium channels that have been characterized, activation time constants range
from 0.1 to 10 ms with threshold potentials from -80 to -20 mV. Inactivation time constants range
from 0.1 to oo (i.e. no inactivation) with threshold potentials from -60 to -20 mV. Time constants
for release from inactivation range from 0.5 ms to 100 ms with threshold potentials from -70 to -40
mV.
25 Choice of cell line and induction of voltage-dependent calcium currents are performed
using the general guidelines and approaches discussed above for sodium channels.
Preferred cell types include those cells that express a minimal level of other ion channels,
such as CHO-K1, CHL, and LTK(-). The transfection and selection of clones expressing ion
channels of interest will generally be performed as described above for sodium ion channels
30 normally in the resting state. Alternatively, a cell line which endogenously expresses the channel
of interest could be used. The labeling and measurement of cells with transmembrane potential
dyes will generally be performed as described for sodium ion channels normally in the resting
state. Alternatively, the cells can be loaded with calcium-sensitive fluorescent dyes such as
Calcium Green, fluo3-AM, or indo-1.
35 In cells with low background currents, strong inward calcium currents can be generated by
driving portions of the membrane negative enough to release the channels from inactivation. Then
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WO 02/08748 PCT/US01/21652
by reversing or releasing the external electric field, the channels are exposed to potentials which
activate the channels and permit calcium current to flow into the cell. The reversal potential for
calcium in most cells is generally +60 to +100 mV, so large voltage changes due to calcium influx
are possible. We can use either membrane-bound voltage-sensitive dyes or intracellular calcium
5 dyes to monitor the activity of the cells. Due to the similarity in properties of calcium and sodium
channels, the same general assay optimization procedures outlined above for sodium channels will
apply to calcium channels.
Typically one would start out with initial conditions using a biphasic square wave kernel of
5 msec per phase and an amplitude of 25 V/cm. The kernel would be repeated at a regular rate of
10 about 20 Hz for a total train duration of about three seconds. One would then optimize the pulse
amplitude, duration, and then frequency. If necessary changes in the pulse shape could also be
explored to determine if these resulted in more efficient electrical stimulation. The optimal
stimulus parameters will yield the maximum cellular stimulation (compared to cells with the
channel blocked, or not present) with the smallest coefficient of variation of the signal among the
15 different test wells, at the lowest electric field strength, and at the lowest duty cycle for passage of
current through the electrodes. After a particular set of parameters is chosen, a titration of staining
concentrations for the voltage sensor dye(s) should be performed as described above, to further
optimize the signal size and coefficient of variation of the responses. These procedures (dye
concentrations, electric field strength, and stimulus duration and frequency) can be iterated to
20 further optimize the signal.
During these experiments, the response will be compared for cells with active channels,
and for cells in which the channels are pharmacologically blocked. If a suitable pharmacological
agent is not available, the blocked state can be emulated with an un-transfected cell line. The
optimal stimulus parameters will yield the smallest coefficient of variation of the difference in
25 signals of the two cell populations.
d) Assay of voltage-dependent chloride channels
Chloride channels are found in the plasma membranes of virtually every cell in the body.
Chloride channels mediate a variety of cellular functions including regulation of transmembrane
potentials and absorption and secretion of ions across epithelial membranes. When present in
30 intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also
regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:1 1 1-122.
Three distinct classes of chloride channels are apparent based on their type of regulation
and structural conformation, Table 3. The first class includes the GAB A and Glycine receptor
super families, the second class includes the CFTR (Cystic fibrosis Transmembrane Conductance
35 Regulator) and the third class includes the voltage regulated chloride channels.
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PCT/US01/21652
Table 3
Chloride Channel Sub-type Summary
Channel Type
Sub-type
Tissue
Distribution
Reference
Ligand gated
GABAa
Receptor family
CNS & PNS
Synapse 21, 189-274 (1995)
Glycine
Receptor family
CNS & PNS
Trends Neurosci. U, 458-461 (1991)
cAMP regulated
CFTR
Epithelial tissues
Science 245, 1066-1073 (1989)
Voltage
regulated
'I
ClC-1
Skeletal Muscle
Nature 354, 301-304 (1991)
ClC-2
Ubiquitous
Nature 356, 57-60(1992)
CIC-Ka
Kidney
J. Biol. Chem. 268, 3821-3824 (1993)
CIC-Kb
Kidney
P.N.A.S. 91, 6943-6947 (1994)
ClC-3
Broad, e.g.
Kidney & Brain
Neuron 12, 597-604 (1994)
ClC-4
Broad, e.g.
Kidney & Brain
Hum. Mol. Genet. 3 547-552 (1994)
ClC-5
Mainly Kidney
J. Biol. Chem. 270, 31172-31177 (1995)
ClC-6
Ubiquitous
FEBS. Lett. 377, 15-20(1995)
ClC-7
Ubiquitous
FEBS. Lett. 377. 15-20(1995)
In contrast to ions like sodium and especially calcium, the electrochemical gradient of
chloride across the plasma membrane is generally not far from equilibrium. Thus, at the resting
potential of cells, the opening of chloride channels will not lead to large excursions of the plasma
5 membrane voltage or dramatic changes in intracellular chloride concentrations. Because electrical
stimulation typically generates symmetrical voltage changes across the cell membrane, no net
chloride flux can be generated unless the conductivity of the channel is non-linear. For a linear
leak conductance, a uniform electric field will drive chloride into the cell on one side and out of the
cell on the other side.
10 Direct electrical stimulation of chloride channels which have non-linear conductance
curves (rectifiers) or voltage-activated gating can generate net ion fluxes, which in turn will cause
detectable transmembrane potential changes. Depending upon the voltage dependence of the
conductance and gating, the transmembrane potential change can be either positive or negative.
For typical chloride channels (that activate at elevated potentials and close at more negative
15 potentials) and for outward rectifiers, chloride will flow into the cell and drive the transmembrane
potential negative. For inward rectifiers, chloride will be driven out of the cell and the
transmembrane potential will be driven positive.
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WO 02/08748 PCT/US01/21652
Due to the small difference between the chloride reversal potential and the resting
transmembrane potential, direct stimulation of a voltage-gated chloride channel may result in
insufficient transmembrane potential changes. Assays for these ion channels can then be
developed using co-expression and electrical stimulation of a sodium or potassium counter-channel
5 in order to produce an inward or outward current. Presence or absence of the chloride current can
then be determined by the absence or presence of a transmembrane potential change when the
counter-channel is electrically stimulated.
Preferred cell types include those cells that express a minimal level of other ion channels,
such as CHO-K1, CHL, and LTK (-). The transfection and selection of clones expressing ion
10 channels of interest will generally be performed as described above for sodium ion channels
normally in the resting state. Alternatively, a cell line which endogenously expresses the channel
of interest (or the counter-channel) could be used. The labeling and measurement of cells with
transmembrane potential dyes will generally be performed as described for sodium ion channels
normally in the resting state.
15 Typically one would start out with initial conditions using a biphasic square wave kernel of
5 msec per phase and amplitude of 25 V/cm. The kernel would be repeated at a regular rate of
about 20 Hz for a total train duration of about three seconds. One would then optimize the pulse
amplitude, duration, and then frequency. If necessary changes in the pulse shape could also be
explored to determine if these resulted in more efficient electrical stimulation. The optimal
20 stimulus parameters will yield the maximum cellular stimulation (compared to cells with the
channel blocked, or not present) with smallest coefficient of variation of the signal among the
different test wells, at the lowest electric field strength, and at the lowest duty cycle for passage of
current through the electrodes. After a particular set of parameters is chosen, a titration of staining
concentrations for the voltage sensor dye(s) should be performed as described above, to further
25 optimize the signal size and coefficient of variation of the responses. These procedures (dye
concentrations, electric field strength, and stimulus duration and frequency) can be iterated to
further optimize the signal.
During these experiments, the response will be compared for cells with active channels,
and for cells in which the channels are pharmacologically blocked. If a suitable pharmacological
30 agent is not available, the blocked state can be emulated with an un-transfected cell line. The
optimal stimulus parameters will yield the smallest coefficient of variation of the difference in
signals of the two cell populations.
e) Assay of ligand dependent channels
The ligand-dependent ion channel family is large and diverse. Ligand-dependent ion
35 channels open in response to the binding of specific molecules. They typically mediate fast
synaptic transmission between neurons, and from neurons to muscle cells. They also mediate slow
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WO 02/08748 PCT/US01/21652
synaptic transmission and control a variety of regulatory mechanisms. Ligand-gated ion channels
are generally only charge-selective; that is, they permit the flow of a range of either anions or
cations but have little specificity. They have enormous variation in their activation, deactivation,
and desensitization kinetics, all of which can vary from submillisecond to second time constants.
5 When the ligand binds to the receptor of the channel, the channel undergoes one or more
conformational changes to activate the channel. If the ligand is removed from the bathing saline,
the bound ligands dissociate and the channel closes. If the ligand remains in the bathing saline,
some channels desensitize by retaining the ligand but moving into a different conformational state
in which the channel is closed. Equilibrium distributions between the activated, deactivated, and
10 desensitized states vary greatly among channels.
In current assay formats, the transmembrane potential of the cells is monitored during an
addition of ligand. The sudden increase in conductance when the channel opens drives the
transmembrane potential towards a new reversal potential. Unfortunately, for many ligand-gated
channels, the new reversal potential is usually within 15 mV of the resting potential. This small
15 change is sufficient to use for signaling within cells, but it makes pharmacological assays difficult.
In an electrical stimulation assay for ligand-gated ion channels, one approach is to co-
express a voltage-gated sodium counter channel with the ligand gated ion channel of interest. This
approach allows us to modulate the transmembrane potential via electrical stimulation. If the test
compounds are added to the cells during or prior to electrical stimulation, the method enables an
20 analysis of whether the ligand gated channel is open or closed. If the ligand-gated channels are
open, the high resting conductance of the cell will suppress the voltage response to electrical
stimulation. If, however, the ligand-gated channels are blocked, the cells will have a large
response to electrical stimulation. The large amount of flexibility in electrical stimulation
parameters should allow us to assay for a large range in resting conductances. This is important in
25 the case of ligand-gated channels, because the resting conductance in the presence of ligand is very
sensitive to the equilibrium desensitization. Accounting for desensitization and variations in
channel expression, we may have resting membrane resistances ranging anywhere from 10 to
10 GQ. With rat brain type Ha sodium channels as the counter channel, we can cover this entire
range. It should also be possible to screen for both agonists and antagonists. By choosing
30 stimulation parameters such that the response is half-size, agonists will reduce the response while
antagonists will increase it. Better screening windows may be obtained by stimulating at higher
(agonist assay) or lower (antagonist assay) frequencies. Note that modulators of the channel
conductance, open time, desensitization, and deactivation will all be detected.
Preferred cell types include those cells that express a minimal level of other ion channels,
35 such as CHO-K1, CHL, and LTK (-). The transfection and selection of clones expressing ion
channels of interest will generally be performed as described above for sodium ion channels
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normally in the resting state. Alternatively, a cell line which endogenously expresses the channel
of interest (or the counter-channel) could be used. The labeling and measurement of cells with
transmembrane potential dyes will generally be performed as described for sodium ion channels
normally in the resting state.
5 Typically one would start out with initial conditions using a biphasic square wave kernel of
5 msec per phase and amplitude of 25 V/cm. The kernel would be repeated at a regular rate of
about 20 Hz for a total train duration of about three seconds. One would then optimize the pulse
amplitude, duration, and then frequency. If necessary changes in the pulse shape could also be
explored to determine if these resulted in more efficient electrical stimulation. The optimal
10 stimulus parameters will yield the maximum cellular stimulation (compared to cells with the
ligand-gated channel blocked, or not present) with smallest coefficient of variation of the signal
among the different test wells, at the lowest electric field strength, and at the lowest duty cycle for
passage of current through the electrodes. After a particular set of parameters is chosen, a titration
of staining concentrations for the voltage sensor dye(s) should be performed as described above, to
15 further optimize the signal size and coefficient of variation of the responses. These procedures
(dye concentrations, electric field strength, and stimulus duration and frequency) can be iterated to
further optimize the signal.
During these experiments, the response will be compared for cells with active channels,
and for cells in which the channels are pharmacologically blocked. If a suitable pharmacological
20 agent is not available, the blocked state can be emulated with an un-transfected cell line. The
optimal stimulus parameters will yield the smallest coefficient of variation of the difference in
signals of the two cell populations.
f) Assay of passive channels
Many channels have slow or no voltage-activated conductance changes. Prime examples
25 are the some of the channels implicated in cystic fibrosis, particularly the cystic fibrosis
transmembrane regulator (CFTR, a chloride channel), the epithelial sodium channel (ENaC) and 4
TM potassium channel family members (Wang et al. Ann. N. Y. Acad. Sci. 868: 286-303, 1999).
A small molecule which acts as an agonist for either of these channels would be a candidate for a
drug which alleviates cystic fibrosis. Currently, there is no convenient workable high throughput
30 screening method for channels of this type.
The proposed assay format for ion channel targets of this type involves a cell expressing
the leak channel of interest in a cell which also expresses a voltage-dependent sodium channel.
The channel of interest is cloned into a cell with a voltage-dependent sodium channel. The
presence of the passive current will suppress the positive response of the sodium channel when the
35 cells are stimulated. Blocking the passive channel will restore the large positive voltage response.
Optimization of the balance of currents will be important in this method. Wild-type CHO cell may
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be useful for this purpose, although a cell with larger sodium currents (either endogenous or
engineered) would be preferable. If the sodium current is too small relative to the potassium
current, the dose-response curve for the passive channel blocker will be shifted towards higher
concentrations. For example, in the extreme case where the passive current is 100 times larger
5 than the sodium current, 99% of the passive channels would have to be blocked in order to get a
50% response from the sodium channels.
Preferred cell types include those cells that express a minimal level of other ion channels,
such as CHOK1, CHL, and LTK (-). The transfection and selection of clones expressing ion
channels of interest will generally be performed as described above for sodium ion channels
10 normally in the resting state. Alternatively, a cell line which endogenously expresses the channel
of interest (or the counter-channel) could be used. The labeling and measurement of cells with
transmembrane potential dyes will generally be performed as described for sodium ion channels
normally in the resting state.
A preferred stimulation protocol uses a biphasic kernel. In general, a series of initial
15 experiments are conducted using a biphasic square wave kernel repeated at a regular rate for a
fixed train duration. The pulse duration varies from about 1 jus to about 1 s, and more preferably
from about 100 \is to about 20 ms. The pulse amplitude varies from 0 V/cm to about 60 V/cm, and
more preferably from 10 V/cm to 50 V/cm. The frequency of stimulation varies between 0 Hz (i.e.
a single pulse) and 100 kHz, and more preferably from 0 Hz to about 1 kHz. The pulse train varies
20 between 0 s (i.e. a single pulse) and about 100 s, and more preferably between 0 s and 10 s.
Typically one would start out with initial conditions using a biphasic square wave kernel of
5 msec per phase and an amplitude of 25 V/cm. The kernel would be repeated at a regular rate of
about 20 Hz for a total train duration of about three seconds. One would then optimize the pulse
amplitude, duration, and then frequency. If necessary changes in the pulse shape could also be
25 explored to determine if these resulted in more efficient electrical stimulation. The optimal
stimulus parameters will yield the maximum cellular stimulation (compared to cells with the
ligand-dependent channel blocked, or not present) with smallest coefficient of variation of the
signal among the different test wells, at the lowest electric field strength, and at the lowest duty
cycle for passage of current through the electrodes. After a particular set of parameters is chosen, a
30 titration of staining concentrations for the voltage sensor dye(s) should be performed as described
above, to further optimize the signal size and coefficient of variation of the responses. These
procedures (dye concentrations, electric field strength, and stimulus duration and frequency) can be
iterated to further optimize the signal.
It should be possible to screen for both agonists and antagonists. By choosing stimulation
35 parameters such that the response is half-maximal, agonists will reduce the response while
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antagonists will increase it. Better screening windows may be obtained by stimulating at higher
(agonist assay) or lower (antagonist assay) frequencies.
During these experiments, the response will be compared for cells with active channels,
and for cells in which the channels are pharmacologically blocked. If a suitable pharmacological
5 agent is not available, the blocked state can be emulated with an un-transfected cell line. The
optimal stimulus parameters will yield the smallest coefficient of variation of the difference in
signals of the two cell populations.
The present invention also includes methods for the quantitative determination of cellular
and ion channel parameters, and for the quantification of the pharmacological effects of test
10 compounds on these parameters using electrical stimulation.
b) Quantitative measurements of membrane resistances
After the electrical stimulus ends, the cell transmembrane potential relaxes to a new resting
potential. In the case of voltage-dependent channel assays, the channels will generally close or
inactivate, and the final resting equilibrium potential will be the same as before the stimulus. In
15 most cases, the charge built up on the membrane capacitance will dissipate exponentially through
the membrane resistance. The membrane time constant is simply the product of the membrane
capacitance and the membrane resistance, x m =R m C m . It can be readily determined by measuring
the membrane capacitance and the membrane time constant.
The average membrane capacitance for cells commonly used in these assays is
20 independent of the exogenous channel, and can easily be measured by patch clamp methods. The
membrane time constant can be readily measured by measuring the rate of decay of the
transmembrane potential and fitting this data to an exponential decay function. Thus by dividing
the membrane time constant by the average membrane capacitance for the given cell type, we can
quantitatively determine the resting or leak membrane resistance.
25 A similar analysis can be made to quantitatively measure the membrane resistance while a
voltage-dependent channel is open. During the electrical stimulation, the transmembrane potential
will also relax approximately exponentially towards a new equilibrium potential. Thus, the
membrane time constant of the voltage change at the beginning of the stimulus constitutes a
measurement of the time-averaged membrane resistance. Using appropriate scaling factors to
30 account for the fraction of the time that the channel is actually open, we can make a quantitative
estimate of the open-channel membrane resistance.
c) Measurement of release from inactivation time constant
Opening an inactivation ion channel requires holding the transmembrane potential below a
threshold for a time on the order of several milliseconds. This release from inactivation has
35 important physiological implications. For example, release from inactivation forces a refractory
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period which prevents back-propagation of action potentials, and limits the maximum firing rates
of neurons. Pharmacological manipulation of this property may be therapeutically relevant
Using repetitive electrical stimulation, we can estimate the average release from
inactivation time. This can be done by using electric field pulses of variable width. When the
5 pulse width falls below the release from inactivation time, fewer channels will be activated and the
transmembrane potential rise in response to the stimulation will drop.
d) Measurement of the open channel time
The open channel time x 0 p en is a function of the inactivation properties of the channel.
We can detect pharmacological manipulation of this parameter in a medium- to high-throughput
10 mode by stimulating at very high frequency. For example, consider an assay for a voltage-
dependent sodium channel using the multiple stimulus method. With a fixed monophasic square
wave stimulus kernel repeated at a steady rate, the voltage response increases as the stimulus
repetition rate increases. This is because the sodium channel spends relatively more time open at
higher frequency. However, if the inter-pulse interval becomes shorter than the open channel time,
15 the activated sodium channels will be driven negative, and thereby deactivated, by the subsequent
stimulus pulse. The stimulation burst frequency at which the response flattens is related to the open
channel time.
e) Electrical stimulation as an extracellular current damn device
In whole-cell recording, current clamp is a mode in which command currents can be driven
20 into the cell while recording the transmembrane potential. Although patch-clamp recording is
extremely precise, it is a very low-throughput technique. At an absolute maximum under perfect
conditions, a highly trained scientist could determine cellular parameters at a rate of about ten cells
per hour. Often, the level of detail obtained with the patch-clamp technique is not necessary for
drug screening, but there is currently no method for exchanging detail for speed. High speed is
25 absolutely crucial for screening large compound libraries.
The electric field stimulation techniques discussed herein permit a new type of current-
clamp electrophysiology which we call extracellular current clamp. Voltage-dependent channels
can be used to drive command currents into cell cultures, allowing determination of several cellular
and channel properties. Extracellular current clamp has a very high throughput, so that it will be
30 possible to obtain high information content of the pharmacological effects of compound libraries
against specific ion channel targets. The pharmacology and physiology of a channel can be studied
directly, or the channel can be used as a current generator for the study of the cell membrane itself
or a second ion channel.
While the ultimate precision of the microscopic parameters obtainable with the
35 extracellular current clamp cannot yet approach the patch-clamp method, we now have the ability
to exchange information content for throughput. That is, the degree of precision at which to make
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measurements can be arbitrarily set. With a single set of stimulus parameters, large libraries can
be screened for potential interesting compounds. A medium throughput secondary screen using a
titration of compound concentrations can be performed on the hits to determine potency and
specificity. Finally, we can determine such therapeutically relevant properties such as use-
5 dependence and mechanism of action by varying the stimulus parameters in the presence of the
compounds. At every stage, the measurements are automatically averaged over many cells, greatly
reducing uncertainties associated with cell-to-cell variability.
There are at least two additional advantages of the extracellular current clamp as compared
to patch-clamp analysis. First, the integrity of the cell membrane is not altered during electric field
10 stimulation. The intracellular fluid is completely replaced with pipette solution during whole-cell
patch clamp recording. Many proteins within the cell, including ion channels, are extremely
sensitive to modulators, intracellular messengers, and the ionic environment. The components of
the cytoplasm are only crudely known, so the soluble components in the intracellular space are
always altered. Therefore, the 'normal' physiological state of the cell is only approximated during
15 whole-cell patch clamp analysis, but remains intact when using extracellular current clamp.
Second, most cells experience dramatic alterations in gene expression and behavior when
in contact with other cells. Because most cells also make gap junctional connections with
neighboring cells, whole-cell patch clamp analysis is only reliable when cells are completely
isolated from each other. Extracellular current clamp can be used on cells independently of their
20 degree of confluence, so the cells may be more physiologically relevant. We can use extracellular
current clamp to find out if there are any effects of cell-cell contact on channel electrophysiology.
Then, in conjunction with gene expression analysis, we can relate these changes to regulatory
components of the cell.
f) Electrical stimulation as an extracellular voltage clamp device
25 In voltage-clamp, the transmembrane potential of the cell is controlled while monitoring
the current flow. Voltage clamp is generally achieved by adding a feedback loop to a current
clamp circuit. In the case of the whole-cell method, this can easily be achieved with the use of two
pipettes simultaneously attached to the same cell. One pipette passes a command current, while
the other senses the voltage. A feedback circuit compares the measured voltage with the command
30 voltage, and adjusts the command current accordingly. Generally, because the cell membrane
resistance is large compared to the access resistance of the pipette, the same pipette can be used to
command the current and measure the voltage. Compared to current clamp, voltage clamp is
generally a more powerful method for electrophysiological analysis. Ion channels are extremely
sensitive to transmembrane potential, so that analysis of data is far more straightforward when
35 dealing with current measurements at a fixed voltage.
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Extracellular current clamp can be converted to a voltage clamp method by adding a
feedback loop between the voltage measurement (the fluorescence of the sensor dye) and the
current generator (the stimulus parameters). In this case, a transmembrane potential dye with
sufficient speed is required. The dye combination CC2-DMPE/DiSBAC6(3) has a submillisecond
5 time constant and should be sufficiently fast to capture all but the fastest cellular events. Based
upon the difference of the command voltage and the transmembrane potential measurements, a
computer will alter the stimulus parameters. The stimulus parameters are related to the current
driven into the cell, so we can determine the time course of the current as a function of the
command voltage. This method should prove useful in determining the mechanism of action of
10 pharmacological agents upon ion channels targets.
g) Assays for intracellular compartments
The stimulation methods described herein can also be used to modulate the transmembrane
potentials of intracellular organelles that have phospholipid membranes, including the
mitochondria and the nucleus. This can be accomplished by first increasing the conductance of the
15 plasma membrane either by electropermeablization or through the addition of ionophores such as
valinomycin or gramicidin A. Then, the intracellular space is no longer insulated from the applied
electric field. This allows an electric field applied to the saline to generate transmembrane
potential changes across the membranes of intracellular organelles. Then, by staining the cells
with dyes which are sensitive to the ion concentration or transmembrane potential, and which are
20 targeted only to the specific organelle membrane of interest, the methods presented herein can be
used to modulate and assay the ion channels of these organelles. Targeting can be achieved, for
example via the use of a naturally fluorescent protein containing suitable subcellular location
signals as are known in the art.
IX. Introduction of exogenous molecules
25 Dielectric breakdown of mammalian cell membranes occurs if the electric potential across
the membrane exceeds about 200 mV (Teissie and Rols, 1993, Biophys. J. 65:409-413). When the
membrane breaks down, pores are formed through the membrane, bridging the intracellular and
extracellular spaces. The number and size of the pores increases with increasing transmembrane
potentials (Kinoshita and Tsong, 1977, Nature 268:438-441). Increasing the electric field strength
30 above about 60 V/cm on typical mammalian cell lines can electropermeablize the cells. At
relatively low fields, small pores are created in the cell membrane which apparently are large
enough to admit small ions, but not large enough to admit molecules as large as DNA (Tsong,
1991, Biophys. J. 60:297-306). These pores totally depolarize the cell, driving the transmembrane
potential to near zero. By electropermeablizing cells and monitoring the transmembrane potential
35 change with a voltage- sensitive dye, the present invention can be used to determine the resting
transmembrane potential of a cell. This will be useful for determining pharmacological
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interactions with cells or ion channels, either as a primary or a secondary screen. For example, in a
compound screen against a voltage-dependent sodium channel, one could perform a multiple
stimulus protocol to determine channel activity. Then, by following with a permeablizing
protocol, one could determine whether or not the cell membrane had a normal resting potential in
5 the presence of the compound.
Additionally, using a highly polarized cell line such as RBL cells, voltage sensitive dyes
could be easily calibrated by electropermeablization. The starting transmembrane potential under
various conditions (for example, various concentrations of extracellular potassium), and the final
transmembrane potential after electropermeablization is zero.
10 Additionally, the size of the pores created by electropermeablization increases as a
function of the applied electric field. Below 50 V/cm, no pores are created. Between about 60
V/cm and 100 V/cm, pores large enough to admit monovalent ions are created. Above around 600
V/cm, pores large enough to admit DNA are created (Tsong, 1991, Biophys. J. 60:297-306).
Thus, this invention can be used to create pores of defined size in the cell membranes, in a high-
15 throughput manner. This could be useful for many applications, including delivery to the
intracellular space of impermeant ions, impermeant test compounds or other modulators, DNA or
RNA for the purpose of transient or stable transfection, and fluorescent or other indicator dyes.
X. Drug Discovery and Screening
a) Drug Screening
20 The present invention provides for the reliable detection of test compounds that modulate
ion channel function that is significantly more versatile and robust than previous assay systems.
Importantly, the present invention provides the ability to modulate the transmembrane potential in
intact cells without the requirement of pharmacological agents, or membrane destruction, and loss
of intracellular contents, as in patching clamping. By providing the ability to externally modulate
25 the transmembrane potential of living cells, the present invention enables a wide variety of ion
channels to be assayed.
Furthermore, this ability to modulate precisely the voltage dependent state of an ion
channel, has important advantages for drug discovery where it provides the opportunity to screen
for compounds that interact preferentially with one state, (i.e. use-dependent blockers). For
30 example, several known therapeutically useful drugs (including anti-arrhythmics, anti-convulsants,
and local anesthetics) are known to function as use-dependent blockers of voltage-dependent
sodium and/or calcium channels. In each case, total blockade of the targeted channel would
typically result in death. Certain conditions, such as chronic pain, arrhythmia, and convulsions
occur when ceils become over-active. These conditions can be alleviated or eliminated by
35 blocking the channels if they begin to open too often. Compounds that are capable of blocking the
channel, but which bind preferentially to the activated or inactivated states(s) rather than the
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resting state(s), can reduce the excitability of muscle and neurons. These drugs are effective
because they do not affect the channel under normal circumstances, but block it only when
necessary to prevent hyper-excitability. However existing methods of analysis that are compatible
with high throughput screening do not provide the ability to routinely control the activation state of
5 the ion channel in real time.
In particular, the present invention provides for a method for screening the effect of a test
compound on an ion channel in a defined functional state within a cell. The method involves
modulating the transmembrane potential of the cell via the use of repetitive electrical stimulation
to cycle the ion channel of interest through its activation cycle and to set the transmembrane
10 potential to a desired level suitable for a specific activation state, or transition between states.
Then, during or after this process a test compound is added to the cell, and the transmembrane
potential is measured.
Typically the results obtained in the presence of the test compound will be compared to a
control sample incubated in the absence of the test compound. Control measurements are usually
15 performed with a sample containing all components and under the same stimulation conditions, as
for the test sample except for the putative drug. Additional control studies can be carried out with
the ion channel in another voltage dependent state to specifically identify state specific test
compounds. Detection of a change in transmembrane potential in the presence of the test agent
relative to the control indicates that the test agent is active and specific on the ion channel in that
20 state, or during the transition from one state to another.
Transmembrane potentials can be also be determined in the presence or absence of a
pharmacologic agent of known activity (i.e., a standard agent) or putative activity (i.e., a test
agent). A difference in transmembrane potentials as detected by the methods disclosed herein
allows one to compare the activity of the test agent to that of the standard agent. It will be
25 recognized that many combinations and permutations of drug screening protocols are known to one
of skill in the art and they may be readily adapted to use with the present inventions disclosed
herein to identify compounds, which affect ion channels and or transmembrane potentials. Use of
the present inventions in combination with all such methods are contemplated by this invention.
In another aspect the present invention includes the use of a second ion channel in
30 conjunction with electrical stimulation methods described herein to set the resting, or stimulated
transmembrane potential to a predefined value thereby providing for the ability to assay a first ion
channel of interest. In one embodiment the second ion channel is a voltage regulated sodium or
calcium channel which enables the generation of sustained positive transmembrane potentials. In
another embodiment the second ion channel is a voltage regulated potassium channel, enabling the
35 generation of negative transmembrane potentials. The use of these second ion channels enables the
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electrical stimulation method to be used to set the transmembrane potential to virtually any
predefined level.
Because this assay format involves two ion channels, modulators of either channel will
affect the voltage response. In this case additional control studies may be carried out with the
5 parental cell line expressing only the second ion channel used to set the transmembrane potential.
Compounds that block the first ion channel can then be re-tested separately to find out if they have
activity against the second ion channel.
Typically the test compounds screened will be present in libraries of related or diverse
compounds. The library can have individual members that are tested individually or in
10 combination, or the library can be a combination of individual members. Such libraries can have
at least two members, preferably greater than about 100 members or greater than about 1,000
members, more preferably greater than about 1 0,000 members, and most preferably greater than
about 100,000 or 1,000,000 members.
b) Selectivity and Toxicology of Candidate Modulators
15 Once identified, candidate modulators can be evaluated for selectivity and toxicological
effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk
Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Patent Nos: 5,196,313 to
Culbreth (issued March 23, 1993) and U.S. Patent No. 5,567,952 to Benet (issued October 22,
1996).
20 For example primary cell lines, or tissue slices can be used to screen for the effect of the
candidate modulator on the response of the ion channel of interest in its native physiological
context. For example, to screen for drugs that exhibit specific, and/or selective effects on heart
cells it may be preferable to use myocytes or other in vitro cell culture model cell lines. In this
case, a primary screen could be completed in a myocyte derived cell line to identify compounds
25 that either shorten, prolong or block electrically-induced action potentials.
The secondary screen would then be designed to identify compounds that exhibit
potentially adverse effects on the body. For example, this can be accomplished by screening for
the effects of the candidate drug on electrically excitable tissues such as heart or neuronal tissues,
or immortalized cell cultures derived from these tissues. These tissues play critical roles within an
30 organism and any undesired effect of the candidate drug on the ability of these tissues to be
electrically stimulated would be predicted to create potential serious side effects when
administered. As a consequence, active compounds that also impaired the ability of these tissues
to function could be eliminated from consideration as a drug candidate at an early stage, or have
medicinal chemistry performed to reduce the side effects.
35 Additional toxicological analysis of candidate modulators can be established by
determining in vitro toxicity towards a cell line, such as a mammalian (preferably human) cell
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line. Candidate modulators can be treated with, for example, tissue extracts, such as preparations of
liver, such as microsomal preparations, to determine increased or decreased toxicological
properties of the chemical after being metabolized by a whole organism, or via their ability to be
degraded via Cytochrome P450 systems as described in commonly owned U.S. Patent Application
5 no. 09/301,525, filed April 28, 1999, U.S. Patent Application No. 09/301,395 filed April 28, 1999
and U.S. Application No. 09/458,927 filed December 10, 1999. The results of these types of
studies are often predictive of toxicological properties of chemicals in animals, such as mammals,
including humans.
The toxicological activity can be measured using reporter genes that are activated during
10 toxicological activity or by cell lysis (see WO 98/13353, published 4/2/98). Preferred reporter
genes produce a fluorescent or luminescent translational product (such as, for example, a Green
Fluorescent Protein (see, for example, U.S. Patent No. 5,625,048 to Tsien et al, issued 4/29/98;
U.S. Patent No. 5,777,079 to Tsien et al, issued 7/7/98; WO 96/23810 to Tsien, published 8/8/96;
WO 97/28261, published 8/7/97; PCT/US97/12410, filed 7/16/97; PCT/US97/14595, filed
15 8/15/97)) or a translational product that can produce a fluorescent or luminescent product (such as,
for example, beta- lactamase (see, for example, U.S. Patent No. 5,741,657 to Tsien, issued 4/21/98,
and WO 96/30540, published 10/3/96)), such as an enzymatic degradation product. Cell lysis can
be detected in the present invention as a reduction in a fluorescence signal from at least one
photon-producing agent within a cell in the presence of at least one photon reducing agent. Such
20 toxicological determinations can be made using prokaryotic or eukaryotic cells, optionally using
toxicological profiling, such as described in PCT/US94/00583, filed 1/21/94 (WO 94/17208),
German Patent No 69406772.5-08, issued 11/25/97; EPC 0680517, issued 11/12/94; U.S. Patent
No. 5,589,337, issued 12/31/96; EPO 651825, issued 1/14/98; and U.S. Patent No. 5,585,232,
issued 12/17/96).
25 Alternatively, or in addition to these in vitro studies, the bioavailability and toxicological
properties of a candidate modulator in an animal model, such as mice, rats, rabbits, or monkeys,
can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug
Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford
(1979), Osweiler, Toxicology , Williams and Wilkins, Baltimore, MD (1995), Yang, Toxicology of
30 Chemical Mixtures; Case Studies, Mechanisms, and Novel Approaches, Academic Press, Inc., San
Diego, CA (1994), Burrell et ah, Toxicology of the Immune System; A Human Approach, Van
Nostrand Reinhld, Co. (1997), Niesink et al., Toxicology; Principles and Applications, CRC Press,
Boca Raton, FL (1996)). Depending on the toxicity, target organ, tissue, locus, and presumptive
mechanism of the candidate modulator, the skilled artisan would not be burdened to determine
35 appropriate doses, LD50 values, routes of administration, and regimes that would be appropriate to
determine the toxicological properties of the candidate modulator. In addition to animal models,
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human clinical trials can be performed following established procedures, such as those set forth by
the United States Food and Drug Administration (USFDA) or equivalents of other governments.
These toxicity studies provide the basis for determining the therapeutic utility of a candidate
modulator in vivo.
5 c) Efficacy of Candidate Modulators
Efficacy of a candidate modulator can be established using several art-recognized methods,
such as in vitro methods, animal models, or human clinical trials (see, Creasey, supra (1979)).
Recognized in vitro models exist for several diseases or conditions. For example, the ability of a
chemical to extend the life-span of HIV-infected cells in vitro is recognized as an acceptable model
10 to identify chemicals expected to be efficacious to treat HIV infection or AIDS (see, Daluge et aL 9
Antimicro. Agents Chemothen 41:1082-1093 (1995)). Furthermore, the ability of cyclosporin A
(CsA) to prevent proliferation of T-cells in vitro has been established as an acceptable model to
identify chemicals expected to be efficacious as immunosuppressants (see, Suthanthiran et aL,
supra, (1996)). For nearly every class of therapeutic, disease, or condition, an acceptable in vitro
15 or animal model is available. Such models exist, for example, for gastro-intestinal disorders,
cancers, cardiology, neurobiology, and immunology. In addition, these in vitro methods can use
tissue extracts, such as preparations of liver, such as microsomal preparations, to provide a reliable
indication of the effects of metabolism on the candidate modulator. Similarly, acceptable animal
models may be used to establish efficacy of chemicals to treat various diseases or conditions. For
20 example, the rabbit knee is an accepted model for testing chemicals for efficacy in treating arthritis
(see, Shaw and Lacy, J. Bone Joint Surg. (Br) 55:197-205 (1973)). Hydrocortisone, which is
approved for use in humans to treat arthritis, is efficacious in this model which confirms the
validity of this model (see, McDonough, Phys. Then 62:835-839 (1982)). When choosing an
appropriate model to determine efficacy of a candidate modulator, the skilled artisan can be guided
25 by the state of the art to choose an appropriate model, dose, and route of administration, regime,
and endpoint and as such would not be unduly burdened.
In addition to animal models, human clinical trials can be used to determine the efficacy of
a candidate modulator in humans. The USFDA, or equivalent governmental agencies, have
established procedures for such studies (see, www.fda.govl
30 d) Selectivity of Candidate Modulators
The in vitro and in vivo methods described above also establish the selectivity of a
candidate modulator. The present invention provides a rapid method of determining the specificity
of the candidate modulator. For example, cell lines containing related ion channel family members
can be used to rapidly profile the selectivity of a test chemical with respect both to its ability to
35 inhibit related ion channels, and their relative ability to modulate different voltage dependent states
of the ion channels. Such a system provides for the first time the ability to rapidly profile large
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numbers of test chemicals in order to systematically evaluate in a simple, miniaturized high
throughput format the ion channel selectivity of a candidate modulator.
e) An identified chemical, modulator, or therapeutic and compositions
The invention includes compositions, such as novel chemicals, and therapeutics identified
5 by at least one method of the present invention as having activity by the operation of methods,
systems or components described herein. Novel chemicals, as used herein, do not include
chemicals already publicly known in the art as of the filing date of this application. Typically, a
chemical would be identified as having activity from using the invention and then its structure
revealed from a proprietary database of chemical structures or determined using analytical
10 techniques such as mass spectroscopy.
One embodiment of the invention is a chemical with useful activity, comprising a chemical
identified by the method described above. Such compositions include small organic molecules,
nucleic acids, peptides and other molecules readily synthesized by techniques available in the art
and developed in the future. For example, the following combinatorial compounds are suitable for
15 screening: peptoids (PCT Publication No. WO 91/19735, 26 Dec. 1991), encoded peptides (PCT
Publication No. WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication WO
92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomeres such as
hydantoins, benzodiazepines and dipeptides (Hobbs DeWitt, S. et ah, Proc. Nat. Acad. Sci. USA
90: 6909-6913 (1993)), vinylogous polypeptides (Hagihara et aL, J. Amer. Chem. Soc. 114: 6568
20 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann, R. et al., J.
Amer. Chem. Soc. 114: 9217-9218 (1992)), analogous organic syntheses of small compound
libraries (Chen, C. et ah, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, C.Y. et
al., Science 261: 1303 (1993)), and/or peptidyl phosphonates (Campbell, D.A. et ah, J. Org. Chem.
59: 658 (1994)). See, generally, Gordon, E. M. et ah, J. Med Chem. 37: 1385 (1994).
25 The present invention also encompasses the identified compositions in a pharmaceutical
composition comprising a pharmaceutically acceptable carrier prepared for storage and subsequent
administration, which have a pharmaceutically effective amount of the products disclosed above in
a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use
are well known in the pharmaceutical art, and are described, for example, in Remington's
30 Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). Preservatives,
stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
For example, sodium benzoate, acsorbic acid and esters of p-hydroxybenzoic acid may be added as
preservatives. In addition, antioxidants and suspending agents may be used.
The compositions of the present invention may be formulated and used as tablets, capsules
35 or elixirs for oral administration; suppositories for rectal administration; sterile solutions,
suspensions for injectable administration; and the like. Injectables can be prepared in conventional
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forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline,
dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the
like. In addition, if desired, the injectable pharmaceutical compositions may contain minor
5 amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the
like. If desired, absorption enhancing preparations (e.g., liposomes), may be utilized.
The pharmaceutically effective amount of the composition required as a dose will depend
on the route of administration, the type of animal being treated, and the physical characteristics of
the specific animal under consideration. The dose can be tailored to achieve a desired effect, but
10 will depend on such factors as weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize. In practicing the methods of the invention, the products
or compositions can be used alone or in combination with one another, or in combination with
other therapeutic or diagnostic agents. These products can be utilized in vivo, ordinarily in a
mammal, preferably in a human, or in vitro. In employing them in vivo, the products or
15 compositions can be administered to the mammal in a variety of ways, including parenterally,
intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally,
employing a variety of dosage forms. Such methods may also be applied to testing chemical
activity in vivo.
As will be readily apparent to one skilled in the art, the useful in vivo dosage to be
20 administered and the particular mode of administration will vary depending upon the age, weight
and mammalian species treated, the particular compounds employed, and the specific use for
which these compounds are employed. The determination of effective dosage levels, that is the
dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art
using routine pharmacological methods. Typically, human clinical applications of products are
25 commenced at lower dosage levels, with dosage level being increased until the desired effect is
achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes
of administration of the compositions identified by the present methods using established
pharmacological methods.
In non-human animal studies, applications of potential products are commenced at higher
30 dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage for the products of the present invention can range broadly
depending upon the desired affects and the therapeutic indication. Typically, dosages may be
between about 10 jag/kg and 100 mg/kg body weight, and preferably between about 100 jig/kg and
10 mg/kg body weight. Administration is preferably oral on a daily basis.
35 The exact formulation, route of administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See e.g., Fingl et ah, in The Pharmacological Basis
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of Therapeutics, 1975). It should be noted that the attending physician would know how to and
when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust treatment to higher levels if the
clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose
5 in the management of the disorder of interest will vary with the severity of the condition to be
treated and to the route of administration. The severity of the condition may, for example, be
evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose
frequency, will also vary according to the age, body weight, and response of the individual patient.
A program comparable to that discussed above may be used in veterinary medicine.
10 Depending on the specific conditions being treated, such agents may be formulated and
administered systemically or locally. Techniques for formulation and administration may be found
in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990).
Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal
administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary
15 injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections.
For injection, the agents of the invention may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration, penetrants appropriate to the
20 barrier to be permeated are used in the formulation. Such penetrants are generally known in the
art. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for
the practice of the invention into dosages suitable for systemic administration is within the scope
of the invention. With proper choice of carrier and suitable manufacturing practice, the
compositions of the present invention, in particular, those formulated as solutions, may be
25 administered parenterally, such as by intravenous injection. The compounds can be formulated
readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for
oral administration. Such carriers enable the compounds of the invention to be formulated as
tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by
a patient to be treated.
30 Agents intended to be administered intracellularly may be administered using techniques
well known to those of ordinary skill in the art. For example, such agents may be encapsulated
into liposomes, then administered as described above. All molecules present in an aqueous
solution at the time of liposome formation are incorporated into the aqueous interior. The
liposomal contents are both protected from the external micro-environment and, because liposomes
35 fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to
their hydrophobicity, small organic molecules may be directly administered intracellularly.
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Pharmaceutical compositions suitable for use in the present invention include compositions
wherein the active ingredients are contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein. In addition to the active ingredients,
5 these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers
comprising excipients and auxiliaries which facilitate processing of the active compounds into
preparations which can be used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical
compositions of the present invention may be manufactured in a manner that is itself known, for
10 example, by means of conventional mixing, dissolving, granulating, dragee-making, levitating,
emulsifying, encapsulating, entrapping, or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the
active compounds in water-soluble form. Additionally, suspensions of the active compounds may
be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles
15 include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase
the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents that increase the
solubility of the compounds to allow for the preparation of highly concentrated solutions.
20 Pharmaceutical preparations for oral use can be obtained by combining the active
compounds with solid excipient, optionally grinding a resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato
25 starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such
as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum arabic, talc,
30 polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added
to the tablets or dragee coatings for identification or to characterize different combinations of
active compound doses. For this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
35 and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to
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characterize different combinations of active compound doses. Such formulations can be made
using methods known in the art (see, for example, U.S. Patent Nos. 5,733,888 (injectable
compositions); 5,726,181 (poorly water soluble compounds); 5,707,641 (therapeutically active
proteins or peptides); 5,667,809 (lipophilic agents); 5,576,012 (solubilizing polymeric agents);
5 5,707,615 (anti-viral formulations); 5,683,676 (particulate medicaments); 5,654,286 (topical
formulations); 5,688,529 (oral suspensions); 5,445,829 (extended release formulations); 5,653,987
(liquid formulations); 5,641,515 (controlled release formulations) and 5,601,845 (spheroid
formulations).
XI. Embodiments of the Invention
10 Some of the embodiments of the present invention are as follows:
A method of characterizing the biological activity of a candidate compound comprising:
exposing one or more cells to said compound;
repetitively exposing said one or more cells to one or more electric fields so as to effect a
controlled change in transmembrane potential of said one or more cells; and
1 5 monitoring, without using a patch clamp, changes in the transmembrane potential of said
one or more cells.
The above method, where the monitoring comprises detecting fluorescence emission from
an area of observation containing said one or more cells.
The above method, where the electric fields are biphasic.
20 The above method, additionally comprising limiting spatial variation in electric field
intensity so as to minimize irreversible cell electroporation.
The above method, where one or more electrical fields cause an ion channel of interest to
cycle between different voltage dependent states.
The above method, where the one or more electrical fields cause an ion channel of interest
25 to open.
The above method, where the one or more electrical fields cause an ion channel of interest
to be released from inactivation.
The above method, where the one or more cells comprise a voltage sensor selected from
the group consisting of a FRET based voltage sensor, an electrochromic transmembrane potential
30 dye, a transmembrane potential redistribution dye, an ion sensitive fluorescent or luminescent
molecule and a radioactive ion.
The above method, where the one or more cells comprise a voltage regulated ion channel.
The above method, where the voltage regulated ion channel is selected from the group
consisting of a potassium channel, a calcium channel, a chloride channel and a sodium channel,
35 The above method, where the electric field exhibits limited spatial variation in intensity in
the area of observation of less than about 25% from a mean intensity in that area.
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The above method, where the one or more electrical fields varies over an area of
observation by no more than about 15 % from the mean electrical field at any one time.
The above method, where the one or more electrical fields varies over an area of
observation by no more than about 5 % from the mean electrical field at any one time.
5 The above method, where the one or more electrical fields comprises stimulation with
either a square wave-form, a sinusoidal wave-form or a saw tooth wave-form.
The above method, where the one or more electrical fields have an amplitude within the
range of about 10 V/cm to about 100 V/cm,
The above method, where the one or more electrical fields have an amplitude within the
10 range of about 20 V/cm to about 80 V/cm.
The above method, where the one or more electrical fields are repeated at a frequency of
stimulation that is greater than or equal to the reciprocal of the transmembrane time constant of
said one or more cells.
The above method, where the one or more electrical fields are repeated at a frequency of
1 5 stimulation within the range of zero to 1kHz.
The above method, where the one or more electrical fields have a pulse duration within the
range of about 100 microseconds to about 20 milliseconds.
The above method, where the transmembrane potential is developed across the plasma
membrane of said one or more cells.
20 A method of assaying the biochemical activity of a compound against a target ion channel
comprising:
selecting a cell line having a normal resting transmembrane potential corresponding to a
selected voltage dependent state of said target ion channel;
expressing said target ion channel in a population of cells of said selected cell line;
25 exposing said population of cells to said compound;
repetitively exposing said population of cells to one or more electric fields so as to effect a
controlled change in transmembrane potential of said one or more cells; and
monitoring changes in the transmembrane potential of said one or more cells.
The above method, where the target ion channel is exogenously expressed in said cell line.
30 The above method, where the cell line is transfected with nucleic acid encoding said target
ion channel.
The above method, where the cell line expresses insignificant levels of other ion channels.
The above method, where the cell line is selected from the group consisting of CHL,
LTK(-),and CHO-K1.
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The above method, where the target ion channel is a sodium channel, and wherein said
population of cells is selected from the group consisting of CHL cells, LTK(-) cells, and CHO-K1
cells.
The above method, where the target ion channel is a sodium channel, and wherein said
5 population of cells is selected from the group consisting of HEK-293 cells, RBL cells, Fll cells,
and HL5 cells.
The above method, where the target ion channel is a potassium channel, and wherein said
population of cells is selected from the group consisting of CHL cells, LTK(-) cells, and CHO-K1
cells.
10 The above method, where the target ion channel is a calcium channel, and wherein said
population of cells is selected from the group consisting of CHL cells, LTK(-) cells, and CHO-K1
cells.
A method of assaying ion channel activity comprising:
exposing at least one cell to a plurality of electric field pulses so as to create a controlled
1 5 change in transmembrane potential and so as to activate an ion channel of interest; and
detecting ion channel activity by detecting one or more changes in transmembrane
potential without using a patch clamp.
The above method, where the at least one cell comprises a voltage sensor selected from the
group consisting of a FRET based voltage sensor, an electrochromic transmembrane potential dye,
20 a transmembrane potential redistribution dye, an ion sensitive fluorescent or luminescent molecule
and a radioactive ion.
The above method, where the voltage sensor comprises a FRET based voltage sensor.
The above method, where the ion channel of interest is a voltage regulated ion channel.
The above method, where the plurality of electric field pulses cause said ion channel of
25 interest to cycle between different voltage dependent states.
The above method, where the at least one cell is an eukaryotic cell.
The above method, where the at least one cell is a non-excitable cell.
The above method, where the at least one cell is a prokaryotic cell.
The above method, where the at least one cell is a tissue culture cell.
30 The above method, where the at least one cell is a primary cell line.
The above method, where the at least one cell is part of an intact living organism.
A method of assaying ion channel activity comprising:
expressing a selected target ion channel in at least one cell;
expressing a selected counter ion channel in said at least one cell;
35 exposing said at least one cell to a plurality of electric field pulses so as to create a
controlled change in transmembrane potential and so as to activate said counter ion channel; and
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monitoring the transmembrane potential of said at least one celL
The above method, where a transmembrane potential change is detected when said ion
channel of interest is blocked.
The above method, where the ion channel of interest comprises a ligand gated ion channel.
5 The above method, where the counter channel comprises a sodium channel.
A method of modifying the transmembrane potential of a cell comprising repetitively
applying biphasic electric field pulses to said cell, wherein said pulses have a maximum amplitude
of less than approximately 90 V/cm, wherein said pulses are applied at a rate of at least about 1 per
second, and wherein the total duration of each pulse is at least about 1 millisecond.
10 The above method, where the maximum amplitude is approximately 20 to 40 V/cm.
The above method, where the pulse duration is approximately 2 to 10 milliseconds per
phase.
The above method, where the pulses are applied at a rate of approximately 20 to 100 pulses
per second.
15 A method of characterizing the biological activity of a candidate compound comprising:
placing one or more cells into an area of observation in a sample well;
exposing said one or more cells to said compound;
repetitively exposing said one or more cells to a series of biphasic electric fields at a rate of
approximately 20 to 100 pulses per second, wherein said electric fields exhibit limited spatial
20 variation in intensity in the area of observation of less than about 25% from a mean intensity in
that area, and wherein said electric fields produce a controlled change in transmembrane potential
of said one or more cells; and
monitoring changes in the transmembrane potential of said one or more cells by detecting
fluorescence emission of a FRET based voltage sensor from an area of observation containing said
25 one or more cells.
The above method, where the one or more electrical fields cause an ion channel of interest
to open.
The above method, where the one or more electrical fields cause an ion channel of interest
to be released from inactivation.
30 The above method, where the one or more cells comprise a voltage regulated ion channel.
The above method, where the voltage regulated ion channel is selected from the group
consisting of a potassium channel, a calcium channel, a chloride channel and a sodium channel.
The above method, where the one or more electrical fields varies over an area of
observation by no more than about 1 5 % from the mean electrical field at any one time.
35 The above method, where the one or more electrical fields varies over an area of
observation by no more than about 5 % from the mean electrical field at any one time.
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The above method, where the one or more electrical fields are selected from a square
wave-form, a sinusoidal wave-form or a saw tooth wave-form.
A high throughput screening system comprising:
a plurality of wells having a high transmittance portion through which cells present in said
5 wells are optically observable in an area of observation;
two electrodes in each of said plurality of wells;
an optical detector configured to detect light emanating from said wells through said high
transmittance portion;
a power supply connected to said electrodes; wherein said power supply and said
10 electrodes are configured to apply a series of electric fields to cells within said area of observation,
said electric fields having a spatial variation of less than about 25% of a mean field intensity within
said area of observation, said electric fields being effective to controllably alter the transmembrane
potential of a portion of said cells;
a data processing unit configured to interpret said light emanating from said wells through
15 said high transmittance portion as ion channel activity resulting from said transmembrane potential
alterations.
The above high throughput screening system, where the pluarality of wells are located in a
multiwell plate.
The above high throughput screening system, where the high transmittance portion is made
20 from a material selected from the group consisting of glass, quartz, cycloolefln, Aclar,
polypropylene, polyethylene and polystyrene.
The above high throughput screening system, where the high transmittance portion
exhibits less fluorescence when excited with UV light in the range of 250 nm to 400 nm than
polystyrene.
25 The above high throughput screening system, where the electrodes are located in a wall of
said plurality of wells.
The above high throughput screening system, where the electrodes are located in a bottom
layer of said plurality of wells.
The above high throughput screening system, where the multiwell plate comprises up to 96
30 wells.
The above high throughput screening system, where the multiwell plate comprises greater
than 96 wells.
The above high throughput screening system, where the multiwell plate comprises greater
than 384 wells.
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The above high throughput screening system, where the electrodes are made of a material
selected from the group consisting of gold, platinum, palladium, chromium, molybdenum, iridium,
tungsten, tantalum and titanium.
The above high throughput screening system, where the multiwell plate comprises
5 optically opaque materials or pigments to reduce the transmission of light.
The above high throughput screening system, where the electrodes are separated by a gap
within the range of about 1 to 4 mm.
The above high throughput screening system, where the electrodes are separated by a gap
within the range of about 0.1 to 1 mm.
10 The above high throughput screening system, where the electrodes are separated by a gap
within the range of about 0.01 to 0.1 mm.
The above high throughput screening system, where the electrodes are charged to create an
electrical field intensity of between 5 to 100 V/cm across said gap, and wherein the total charge
transferred across the surface area of the electrically conductive material, in fluidic connection
15 with the interior of the well is less than or equal to lOOjuC/mm 2 .
The above high throughput screening system, where the plurality of wells further comprise
an insulator orientated and configured so as to create an area of observation within said well in
which the electrical field intensity varies by no more than 10 % from the mean electrical field
intensity when said at least two strips of electrically conductive material are charged to create an
20 electrical field intensity of between 5 to 100 V/cm across said gap, and wherein the total charge
transferred across the surface area of the electrically conductive material, in fluidic connection
with the interior of the well is less than or equal to lOOjuC/mm^ .
The above high throughput screening system, where the plurality of wells further comprise
at least two satellite electrical conductors.
25 A high throughput screening system comprising:
sample wells;
liquid handling stations for adding reagents and/or cells to said sample wells; and
means for controlling the transmembrane potential of cells in said sample wells so as to
selectively cause ion channel activity.
30 means for optically monitoring changes in said transmembrane potential.
The above high throughput screening system, where the means comprises electrodes
configured to create an electric field having a spatial variation of less than about 25% of a mean
field intensity within an area of observation.
The above high throughput screening system, where the means for controlling the
35 transmembrane potential comprise an electrode array assembly.
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The above high throughput screening system, where the electrode assembly array
comprises 8 electrode assemblies.
The above high throughput screening system, where the electrode assembly array
comprises 96 electrode assemblies.
5 The above high throughput screening system, where the electrode assembly array
comprises greater than 96 electrode asemblies.
The above high throughput screening system, where the system further comprises means
for retractably moving said electrode assembly into and out of the wells of a multiwell plate.
The above high throughput screening system, where the means for controlling the
10 transmembrane potential comprises electrical conductors with two substantially parallel planar
surfaces.
The above high throughput screening system, where the electrical conductors are separated
by a gap within the range of 1 to 4 mm.
The above high throughput screening system, where the electrical conductors are separated
15 by a gap within the range of 0.1 to 1 mm.
The above high throughput screening system, where the electrical conductors further
comprise a first insulator.
The above high throughput screening system, where the first insulator comprises two
planar surfaces orientated perpendicular to said substantially parallel planar surfaces of said
20 electrical conductors and substantially parallel with respect to each other.
The above high throughput screening system, where the electrical conductors further
comprise a second insulator attached to said at least two electrical conductors, wherein said second
insulator is interposed in said gap between said at least two electrical conductors to define the
depth of said aqueous solution between said at least two electrical conductors.
25 The above high throughput screening system, where the first insulator is composed of a
low fluorescence material, wherein said low fluorescence material exhibits less fluorescence when
excited with UV light in the range 250 nm to 400 nm than polystyrene of comparable size.
The above high throughput screening system, where the second insulator is composed of a
low fluorescence material, wherein said low fluorescence material exhibits less fluorescence when
30 excited with UV light in the range 250 nm to 400 nm than polystyrene of comparable size.
The above high throughput screening system, where the first insulator comprises an
insulator selected from the group consisting of plastic, glass and ceramic.
The above high throughput screening system, where the plastic is selected from the group
consisting of nylon, polystyrene, Teflon (tetrafluoroethylene), polypropylene, polyethylene,
35 polyvinyl chloride, and cycloolefin.
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. The above high throughput screening system, where the electrical conductors comprise a
conductor selected from the group consisting of gold, platinum, titanium, tungsten, molybdenum,
iridium, vandium, Nb, Ta, stainless steel and graphite.
The above high throughput screening system, where the electrical conductors comprise a
5 surface treatment to reduce electrolysis.
The above high throughput screening system, where the surface treatment to reduce
electrolysis comprises platinum black, gold black, iridium/iridium oxide, titanium/titanium nitride
or polypyrrole films.
The above high throughput screening system, where the electrical field intensity varies by
10 no more than 10 % from the mean electrical field intensity when said at least two electrical
conductors are charged to create an electrical field intensity of between 5 to 100 V/cm across said
gap, wherein the total charge transferred across the surface area of the electrical conductors in
contact with said aqueous solution is less than or equal to 100 DC/mm2.
The above high throughput screening system, where the electrical field intensity varies by
15 no more than 5% from the mean electrical field intensity when said at least two electrical
conductors are charged to create an electrical field intensity of between 5 to 100 V/cm across said
gap, wherein the total charge transferred across the surface area of the electrical conductors in
contact with said aqueous solution is less than or equal to 100 jnC/mm 2 .
A method of screening a plurality of drug candidate compounds against a target ion
20 channel comprising:
expressing said target ion channel in a population of host cells;
placing a plurality of said host cells into each of a plurality of sample wells;
adding a candidate drug compound to at least one of said plurality of sample wells; and
modulating the transmembrane potential of host cells in said plurality of sample wells with
25 a repetitive application of electric fields so as to set said transmembrane potential to a level
corresponding to a pre-selected voltage dependent state of said target ion channel.
The above method, additionally comprising selecting a host cell line having a normal
resting transmembrane potential corresponding to a second pre-selected voltage dependent state of
said target ion channel.
30 The above method, where the electric fields are biphasic.
The above method, where electric fields cause an ion channel of interest to cycle between
different voltage dependent states.
The above method, where the electric fields cause an ion channel of interest to open.
The above method, where the electric fields cause an ion channel of interest to be released
35 from inactivation.
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The above method, where the one or more cells comprise a voltage sensor selected from
the group consisting of a FRET based voltage sensor, an electrochromic transmembrane potential
dye, a transmembrane potential redistribution dye, an ion sensitive fluorescent or luminescent
molecule and a radioactive ion.
5 The above method, where the target ion channel is selected from the group consisting of a
potassium channel, a calcium channel, a chloride channel and a sodium channel.
The above method, where the one or more electrical fields comprises stimulation with
either a square wave-form, a sinusoidal wave-form or a saw tooth wave-form.
The above method, where the one or more electrical fields have an amplitude within the
10 range of about 10 V/cm to about 100 V/cm.
The above method, where the one or more electrical fields have an amplitude within the
range of about 20 V/cm to about 80 V/cm.
An assay plate and electrode assembly comprising at least one sample well having
electrodes placed therein, wherein said electrodes are positioned with respect to the bottom surface
15 of the well to provide an electric field adjacent to said bottom surface that varies by less than about
10% from a mean field intensity over at least about 20% of the surface area of said bottom surface.
The above assembly, where the electrodes comprise plate electrodes extending down into
said well such that bottom ends of said electrodes are adjacent to but not in contact with said
bottom surface.
20 The above assembly, comprising two electrodes per sample well.
The above assembly, comprising more than two electrodes per sample well.
The above assembly, where the electrodes are plated onto said bottom surface of said well.
The above assembly, where the bottom surface comprises a high optical transmittance
portion.
25 The above assembly, where the high transmittance portion is made from a material
selected from the group consisting of glass, quartz, cycloolefin, Aclar, polypropylene, polyethylene
and polystyrene.
The above assembly, where the high transmittance portion exhibits less fluorescence when
excited with UV light in the range of 250 nm to 400 nm than polystyrene.
30 The above assembly, where the electrodes are located in a wall of said plurality of wells.
The above assembly, where the plate comprises up to 96 wells.
The above assembly, where the plate comprises greater than 96 wells.
The above assembly, where the plate comprises greater than 384 wells.
The above assembly, where the electrodes are made of a material selected from the group
35 consisting of gold, platinum, palladium, chromium, molybdenum, iridium, tungsten, tantalum and
titanium.
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The above assembly, where the electrodes are separated by a gap within the range of about
1 to 4 mm.
The above assembly, where the electrodes are separated by a gap within the range of about
0.1 to 1 mm.
5 The above assembly, where the electrodes are separated by a gap within the range of about
0.01 to 0.1 mm.
A bottom panel for a multi-well plate comprising:
at least one row of high transmittance regions with positions corresponding to well
locations;
10 a first strip of conductive material extending along said row and overlapping a first portion
of said well locations; and
a second strip of conductive material extending along said row and overlapping a second
portion of said well locations.
The above bottom panel, additionally comprising a first electrical contact proximate to an
15 end of said first strip and a second electrical contact proximate to an end of said second strip.
An assay apparatus comprising:
a sample well;
a first pair of electrodes positioned within said sample well;
at least one additional satellite electrode positioned within said sample well.
20 The above assay apparatus, where the at least one additional satellite electrode comprises
second and third pairs of electrodes.
The above assay apparatus, where the satellite electordes are charged to a potential less
than that of the first pair of electrodes.
The above assay apparatus, where the electrodes are positioned with respect to the bottom
25 surface of the well to provide an electric field adjacent to said bottom surface that varies by less
than about 10% from a mean field intensity over at least about 20% of the surface area of said
bottom surface.
EXAMPLES
The invention may be better understood with reference to the accompanying examples, which
30 are intended for purposes of illustration only and should not be construed as in any sense limiting the
scope of the invention as defined in the claims appended hereto.
Example 1 : Analysis of the electrical field uniformity of parallel plate electrodes in standard
round wells.
To analyze the effect of various electrode, and well designs, a series of two-dimensional
35 numerical simulations of the electric fields were produced using the software analysis package
Quickfield™ 4.1, (Student's Version, Tera Analysis, http^/www.tera-analvsis.com") . This software
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package creates coarse-grained mesh type electrical field intensity maps by solving Poisson's
equation with a finite-element analysis method in two dimensions. For the purposes of this
analysis, the fringing effects due to the gap between the bottom of the electrode and the bottom of
the well were ignored, and the voltage drops from the electrodes to the saline were also assumed to
5 be negligible. The spatial resolution of the modeling is approximately 0.5 mm.
FIG. 7A shows the results of the simulation using 4 mm wide parallel plate electrodes
(710) with a 4 mm gap with a standard electrical potential difference of 2V located in a standard
circular 96-well. In this figure, the outer circle (700) is the edge of the well, the two vertical lines
(710) are the electrodes, and the dashed circle in the middle (720) is the area of observation. The
10 gray area (740) is the area in which the electric field remains within ±10% of the mean field in the
area of observation. In the white area (730), the field is less than 10% of the mean, and in the
black area (750), the field is more than 10% greater than the mean field. Within the area of
observation, the standard deviation of the field strength is 2% of the mean, and the difference
between the maximum and minimum fields is 10% of the mean. Thus, this geometry satisfies the
15 stated requirements for field uniformity for use in the present invention.
Example 2: Analysis of the electrical field uniformity of pin electrodes in standard round
wells.
To determine the predicted field uniformity for two 1 .0 mm diameter round pin electrodes
placed in a standard 6.2 mm diameter well, separated by a distance of 4.0 mm, simulations were
20 completed with the same conditions and assumptions as described in Example 1.
In FIG, 7B, the outer solid circle (705) is the edge of the well, the two smaller circles
(715) are the electrodes, and the dashed circle in the middle is the area of observation. The gray
area (745) is the area in which the electric field remains within ±10% of the mean field in the area
of observation. In the white area (735), the field is less than 10% of the mean, and in the black
25 area (755), the field is more than 10% greater than the mean field. Within the area of observation
(725), the standard deviation of the field strength is 15% of the mean, and the difference between
the maximum and minimum fields is 87% of the mean. Thus, this geometry does not create
uniform electrical fields and as a consequence is not suitable for use with the present invention.
Example 3: Analysis of the electrical field uniformity of parallel plate electrodes in square
30 wells
FIG. 8A shows a simulation of the field profile for two 6 mm wide parallel plate
electrodes with a 4 mm gap in a 6.2 mm square well. In this figure, the outer square (800) is the
edge of the well. The two vertical lines (810) are the electrodes. The dashed circle in the middle
(820) is the area of observation. Of particular note is that the electric field scale for FIG. 8 has
35 been greatly amplified compared to FIG. 7 to provide contrast for the variations in electrical field
intensity. The gray area (840) is the area in which the electric field remains within ±1% of the
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mean field in the area of observation. In the white area (830), the field is less than 1% of the mean.
In this simulation, at no point is the field more than 1% greater than the mean field. Within the
area of observation, the standard deviation of the field strength is 0.02% of the mean, and the
difference between the maximum and minimum fields is 0.12% of the mean. Thus, this geometry
5 greatly improves the field uniformity.
The results of the simulation indicate that the primary source of field non-uniformity in the
parallel plate system shown in FIG. 7 A derives from the rounded walls of the well. In a standard
well with a circular cross section, the current density will spread out and then contract as it passes
from one electrode to the other, and this spreading generates non-uniformity. This can be
10 corrected by using multiwell plates with square wells.
Example 4: Analysis of the effect of the addition of insulator elements to mask off rounded
areas of the wells.
FIG. 8B shows a simulation of the field profile for two 4 mm wide parallel plate
electrodes with a 4 mm gap in a 6.2 mm diameter round well using the standard conditions and
15 analysis procedures as described in Example 1. Insulators are attached to the electrodes to mask
off the rounded areas of the well between the electrodes, as shown in FIG. 9A. In FIG. 8B, the
outer circle (802) is the edge of the well. The two vertical lines (812) are the electrodes. The
dashed circle in the middle (822) is the area of observation. The cross-hatched areas (862) are
insulators attached to the electrodes. The gray area (842) is the area in which the electric field
20 remains within ±1% of the mean field in the area of observation. In the white area (832), the field
is less than 1% of the mean. In this simulation, at no point is the field more than 1% greater than
the mean field. Within the area of observation, the standard deviation of the field strength is 0.2%
of the mean, and the difference between the maximum and minimum fields is 1.0% of the mean.
Thus, this geometry greatly improves the field uniformity over the case where no insulator is used,
25 but not as much as in the case of square wells.
The results demonstrate that the field uniformity in standard round well plates can be
greatly increased by filling the area outside of the area defined by the electrodes with an insulating
material. In practice inert plastics such as nylon, tetrafluoroethylene, polycarbonate, or any other
non-porous polymer, or glass, could be used as the insulator material, provided that they are
30 relatively stable to aqueous solutions, easily fabricated and preferably non-fluorescent. The
insulator would typically be attached to the electrode, and would not obscure any of the area
defined by the electrodes.
Example 5: Analysis of the effect of satellite electrodes on field uniformity
To test whether it is possible to compensate for the loss of current into the curved edge of
35 the well via the use of satellite electrodes, simulations were carried out at a variety of electrode
geometries. FIG. 9B shows one possible embodiment of this concept, and FIG. 8C shows the
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electric field profile when this geometry is analyzed using Quickfield™ as described in Example
1. In this example, two extra pairs of 0.7 mm wide parallel plate electrodes were placed with a 2
mm gap. These electrodes are outside of the area of observation, and are maintained at half the
potentials of their parent electrodes.
5 In FIG. 8C, the outer solid circle (804) is the edge of the well. The two long solid vertical
lines (814) are the parent electrodes, and the four shorter solid vertical lines (816) are the satellite
electrodes. The dashed circle in the middle (824) is the area of observation. The gray area (844) is
the area in which the electric field remains within ±1% of the mean field in the area of observation.
In the white area (834), the field is less than 1% of the mean, and in the black area (854), the field
10 is more than 1% greater than the mean field. Within the area of observation, the standard deviation
of the field strength is 0.2% of the mean, and the difference between the maximum and minimum
fields is 1.2% of the mean. Thus, this geometry greatly improves the field uniformity over the case
where no insulator is used, but not as much as in the case of square wells.
This example demonstrated the use of four satellite electrodes in a specific configuration.
15 By adding more satellite electrodes outside of the area of observation, and by properly assigning
their potentials as a function of the potentials applied to the parent electrodes, the electric field
uniformity can, in principle, be improved to arbitrary precision.
For example in a round well configuration, field uniformity in the center area of
observation can be improved by subdividing the parallel plate electrodes into several pieces
20 separated by thin insulating dividers, as depicted in FIG. 9D. The potential applied to each
electrode (expressed as a fraction of the potential applied to the central most piece) can be
individually tuned to maximize the field uniformity in the area of observation.
This concept can be expanded to allow the use of non-parallel dipper electrodes, which
have several vertical conducting stripes, each of which is independently controlled.
25 Example 6: Analysis of the effect of the meniscus on electrical field uniformity
The meniscus created by dipper electrodes when inserted into a well generates variations
of saline depth of around 10% across the area of observation. This in turn generates variations in
the electric field of around 1 0% across the area of observation. These variations exist even if the
electrode design is predicted to create perfect field uniformity. Thus, eliminating the meniscus
30 effect will improve the actual field uniformity. One possible method to accomplish this is to add
an insulating barrier between the electrodes. FIG. 9C depicts one such embodiment, wherein the
insulating barrier is used to create a flat top surface for the liquid in the well. The bottom of this
barrier, when the electrodes are inserted into the well, would be designed to sit approximately 2.5
mm above the bottom of the well. Thus, the barrier would be partially immersed in saline, and its
35 bottom surface would define the top of the conductive chamber to be flat and perpendicular to the
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electrode surfaces. In this way, the electric field would not be perturbed by irregularities in the
surfaces of the conductive volume.
Example 7: Fabrication of dipper electrode electrical stimulator
In one embodiment of the electrical stimulator the device is comprised of a self-locating
5 frame that positions the dipper electrodes into the array of wells in a 96 well multiwell plate format
(FIG. 1). FIG. 1 depicts the inserted position of the electrode array. In this example, the electrical
stimulation device can be assembled from three functional parts. The first part is the positioning
frame (40) that locates the device relative to the plate wells. This frame is made of metal and is a
snug fit to the multiwell plate. This frame serves as the locating base for the second functional part
10 of the system, the retractor mechanism.
The retractor system consists of shoulder bolts (70) and return springs (not visible). The
springs are wrapped around the shoulder bolts, and press against the positioning frame (40) and the
bottom of the insulating cover (90). The return springs hold the electrode assembly in the retracted
position until the electrodes are lowered into the plate wells. The retractor mechanism locates the
15 third functional part of the system, the electrode array.
The third functional part of the system is the electrode array. The electrode array is made
up of eight pairs of identical electrode combs (10). The electrode combs are made of stainless steel
and are precision laser cut to avoid distortion. Each comb has eight tabs of sufficient width to
nearly span the diameter of the multiwell plate wells. The backbone of the comb forms the
20 electrical connection to the tabs (50). Two of these combs form the electrode pairs that are
inserted into a column of eight wells. The combs are held in position relative to each other by an
insulating precision drilled plate (30) that locates the electrodes relative to the positioning frame.
Insulating spacers (20) maintain electrode separation and index the combs to the drilled plate by
means of a pinned interface. A second set of spacers (25) ensures precise positioning of the
25 electrodes (10) relative to the plate (30). Alignment shafts (15) are inserted through alignment
holes in the spacers (20) and the electrode combs (10) for additional stability. The combs and
spacers are held in place against the drilled plate by an insulating cover (90).
The device may be used manually by placing the device on the multiwell plate and
pressing down on the electrode assembly to lower the electrodes into the wells. When the
30 electrodes are fully extended, a pair of locking bars (60) is inserted to keep the electrodes extended
into the wells. Alternatively the electrode array can be automatically inserted and retracted in to
the wells via standard mechanical or robotic control systems known in the art.
FIG. 3 shows a block diagram of the major electrical and optical components. Electrical
stimuli were created via a high-power amplifier (320), driven by a pair of digital function
35 generators (380 and 310). In one embodiment the switch (330) was a National Instruments (Austin
TX) ER-16 controlled by a National Instruments PC-DIO 24 digital input/output card on board the
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VIPR™ reader controller computer (300). The switch (330) allowed defined wells within a 96-
well plate to be electrically stimulated with any given time protocol. In this case, a single column
of eight wells was stimulated simultaneously. The amplifier (320) was built using the APEX PA93
chip (Apex Microtechnology Corp, Tucson, AZ) following a circuit provided by the manufacturer.
5 The amplifier had the following specifications: ±100V DC in, 100 G£2 input impedance, 20X
voltage gain, ±90 V out, ±3 A out, 10 Q, output impedance. The function generators were Tektronix
(Beaverton, OR) model AFG310. The first function generator (380) was triggered by the VIPR™
reader software when the stimulus pulse train was required to begin, and produced a train of TTL
pulses to trigger the second function generator (310). The second function generator was
1 0 programmed with the stimulus waveform kernel.
Example 8: Voltage Dependence of Electrical Stimulation
Wild type Chinese hamster ovary cells (CHO cells) endogenously express a voltage-
dependent sodium channel and can be conveniently used to validate and optimize electrical
stimulation parameters. Besides this sodium channel, these cells appear to have gap-junctional
1 5 connections between adjacent cells and a very small (-20 pA) voltage-dependent outward current.
The voltage dependent sodium channel in these cells (hereafter referred to as NaVl) has
electrophysiological characteristics similar to rat brain type Ha sodium channels. Analysis of the
current / voltage characteristics of this channel via standard electrophysiology reveals that typical
wild type CHO cells have an average peak current of 100 pA per cell at -20 mV. This corresponds
20 to a membrane resistivity (7?N a ) of about 800 M£X Assuming a single-channel conductance of 10
pS, this suggests that there are only -125 sodium channels per cell. In our hands, CHO cells
typically exhibit a resting transmembrane potential (R m ) of about -35 mV, a resting membrane
resistance >10 GQ, and a cell membrane capacitance (C m ) of 15 pF.
To test the voltage dependency of electrical stimulation, wild type CHO cells were seeded
25 into 96 well microtiter plates and incubated in growth medium for 24-48 hours. They were then
rinsed with bath solution 1 and stained for 30 minutes each with 10 uM CC2-DMPE (coumarin),
then 3 uM DiSBAC2(3) (ethyl oxonol as described in Appendix Al). A stimulator assembly with
96 pairs of stainless steel electrodes (4 mm wide, 4 mm gap) was placed atop the assay plate, as
described in Example 7. The electrodes were lowered into the saline covering the cells and
30 remained 0.5 mm from the bottom of the well. Ratiometric fluorescence measurements were made
during electrical stimulation using a VIPR™ reader as described above, and the data were analyzed
according to the procedures in Appendix A2. At any one time, only one column of eight wells was
assayed; the remaining wells received no excitation light or electrical stimulation. After each
plate was assayed, the electrodes were thoroughly rinsed with distilled water and dried with
35 compressed air, to prevent cross-contamination between plates.
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To determine the transmembrane potential changes occurring in the cells as a result of
electrical stimulation, multiwell plates containing the cells were analyzed in a VIPR™ reader. The
cells in a 3 mm diameter area of observation located midway between the electrodes were excited
with light at 400±7.5 nm. The light was generated by a 300 W xenon arc lamp, and passed through
5 a pair of a pair of dielectric interference band-pass filters to select the correct excitation
wavelength. Light was directed to and from the cells via a trifurcated fiber optic cable, with one
cable for excitation light and two for fluorescence emission. Simultaneous measurements of blue
(460±20 nm) and orange (580±30 nm) signals were recorded from each well at 50 Hz, digitized
and stored on a computer. Initial assays were 15 seconds long, and consisted of a 6 second
10 stimulation of repetitive (90 Hz repetition rate) biphasic (5 ms/phase) square-wave stimulation
beginning at 2 seconds at the electrical amplitudes shown. For two seconds before and seven
seconds after the stimulation burst, no current passed through the electrodes. FIG. 10 shows the
ratiometric responses at various field strengths up to 32 V/cm. In this case the apparent rise time
of the recorded response is limited by the response time of the DiSBAC2(3) that has a response
15 time constant of around 1 second. Below pulse amplitudes of 10 V/cm, no response is detectable.
Above 20 V/cm, the response is robust and increases only slightly as the voltage is further
increased up to 32 V/cm. As shown in FIG. 11, at higher voltages, the peak response (measured
after about 5 seconds) shows only further small increases in response. The data in FIG. 11 can be
fitted to a Boltzman function, which had a midpoint at 18.0 V/cm with a 2.0 V/cm width. The
20 sharpness of the onset and the flatness of the response at high fields are strongly suggestive of a
threshold phenomenon. The electric field at which the response is half maximal (18 V/cm)
corresponds to approximately ±30 mV deviations in transmembrane potential at the extreme edges
of the cells, using formulas previously published (Equation 1, see also Tsong, 1991, Biophys. J.
60:297-306; and assuming an average diameter of the cells of 30 jum). It is therefore quantitatively
25 consistent with the stimulation mechanism described above for voltage-gated sodium channels
normally in the inactivated state.
High intensity electrical fields can result in electroporation of the cell membranes resulting
in large relatively non-specific changes in transmembrane potential (Tsong, 1991, Biophys. J.
60:297-306). To establish whether or not this is also a major factor in the responses of the cells to
30 lower electrical field intensities used here, experiments were conducted with the sodium channel
specific toxin tetrodotoxin (TTX). If the effects of electrical stimulation can be blocked by the
toxin, this would suggest that the effect of electrical stimulation is primarily mediated by the
activation of sodium channels. The results of this experiment are shown in FIG. 12. The data was
obtained with electrical field strength of 33 V/cm and demonstrate that TTX was able to
35 completely block the effect of electrical stimulation with typical pharmacological characteristics
consistent with the blockage of sodium channels. The EC50 from the fit to this data is 9 nM,
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similar to the reported value for TTX in rat brain type Ha (8 nM, West et al., 1992, Neuron 8: 59-
70). The fact that this signal is blocked by TTX with normal pharmacology is strong evidence that
the signal generated via electrical stimulation is almost entirely due to NaVl.
Example 9: Variation of Cellular Response to Changes in Stimulus Pulse Width and Frequency
5 To examine the behavior of the cellular response as the stimulus pulse width and frequency
were varied, experiments were carried out using wild type CHO cells as described in Example 8
above at a constant field strength of 25 V/cm, while varying the pulse duration and frequency.
The results are displayed in FIG. 13. Each data point represents the average of eight wells
stimulated at the same time from experiments derived from five separate plates of wild-type CHO
10 cells. The results show generally that as the frequency of stimulation increases the magnitude of
the response increases. One would predict that this effect should eventually saturate as the
transmembrane potential is driven to the sodium reversal potential (V]^ a ). In this case this does
not occur because the sodium channel density is too low.
Increasing the pulse duration results in higher relative degrees of electrical stimulation at
15 lower stimulation frequencies up to about 10 ms, beyond which further increases are less
pronounced. Very small pulse durations (less than 1 ms) also limit the response, apparently
because the channels are not effectively released from inactivation. To efficiently induce large
cellular responses, the best stimulation parameters are typically in the range in which the pulse
duration is greater than, or equal to the time constant for recovery for inactivation, and sufficiently
20 short so that the frequency of stimulation is greater than the membrane time constant. Additionally
the optimal frequency of stimulation is typically less than the reciprocal of the average channel
open time.
These experiments demonstrate that electrical stimulation can be successfully used even in
cells that express even relatively low levels of voltage dependent channels, and can be successfully
25 completed under conditions that do not lead to significant electroporation or cell death. These
experiments also demonstrate methods by which stimulus pulse duration and repetition frequency
can be optimized to produce responses of a desired size.
Example 10: Analysis of CHO cells expressing an exogenous sodium channel
Chinese hamster ovary cells were stably transfected with a plasmid encoding a voltage
30 dependent sodium channel (hereinafter referred to as NaV2) as described in section VI. Whole-
cell patch clamp analysis was used to confirm the electrophysiological and pharmacological
properties of this channel prior to analysis via electrical stimulation. The peak transient sodium
current at -20 mV was measured to be 600 ± 300 pA (N=5), with an average cell membrane
capacitance of 15 ± 5 pF. The resting cell membrane resistance was too large to measure
35 accurately (Rl > 10 GO). The resting transmembrane potential was -3 1 ± 3 mV.
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To determine the threshold electric field for stimulation, cells stably expressing the sodium
channel were plated in 96-well plates and stained according to the protocol in Appendix Al. The
electrical stimulation protocol involved a 20 Hz, 3 second burst of biphasic (5 ms/phase) stimuli
with variable field strength using the electrical stimulator described in Example 7.
5 FIG. 14 shows representative time traces at various field strengths (each curve is the
average of eight wells). At low field strengths, there is no detectable cellular response, suggesting
that the average transmembrane potential changes less than about 1 mV. Between 35 and 90
V/cm, the response is stereotyped, with a fixed shape and amplitude. Above 90 V/cm, the peak
response stays relatively constant, but the response decay time after the stimulus is removed
10 becomes considerably extended.
Consistent with the experiments shown in Example 8, the response induced by electrical
field strengths up to 85 V/cm could be inhibited by TTX whereas the response obtained from cells
stimulated above 90 V/cm could not (data not shown). Therefore we conclude that the fast
response is due to the sodium-channel-opening mechanism outlined above, while the slow
15 response is mainly caused by electropermeablization of the membrane by the electrical field.
This effect is more easily seen by comparing the behavior of the fast response (4 seconds
after stimulation) and the slow response (ten seconds after stimulation) with increasing field
strength. This data is shown in FIG. 15. Fitting the fast response to a Boltzman function, the
midpoint of the early response was at E5o=26 V/cm, with a width of AE=3.5 V/cm. The response
20 was independent of field strength between 40 and 80 V/cm, with a slight increase when
electropermeablization sets in above 90 V/cm.
The slower response due to permeablization was first detectable at 90 V/cm, and is itself of
potential use in some applications. For example, permeablization can be used for resetting the
transmembrane potential to zero, or if the permeablization is selective for a specific ion, for
25 resetting the transmembrane potential to the equilibrium value for that ion. This could be useful,
for example, in an assay for a channel that sets the transmembrane potential. Examples include
potassium and chloride leak channels, potassium inward rectifiers, and low-voltage activated
voltage-gated potassium channels.
These results are consistent with published studies in which electropermeablization begins
30 with a threshold transmembrane potential of around ±200 mV, independent of cell type (Teissie
and Rols, 1993, Biophys. J. 65:409-413). Based on formulae reported in that article and widely
accepted in the literature, CHO cells with an average diameter of 30 jum will experience ±200 mV
transmembrane potential changes when exposed to a 90 V/cm extracellular electric field.
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Example 1 1 : Determination of the effective release from inactivation time and the effective
open-channel sodium conductance.
To make quantitative estimates of the effective release from inactivation time and open
channel conductance, but without being bound to any specific mechanism of action, the following
5 theory was developed for experimental verification.
After opening, the sodium channels inactivate with a voltage-dependent time constant of
order 1 millisecond. Because the current passed by the open sodium channels is strongly voltage-
and time-dependent, it is not possible to easily generate an analytical expression for the voltage
change after a single stimulation. However by making some simplifying approximations, we can
1 0 model average idealized responses to create a testable theory. For the purposes here, we assume
that upon opening, the sodium channels behave as a linear conductance above V\ = -40 mV with a
reversal potential at = +60 mV. The conductance g]\j a is determined as the maximal current
obtained at —20 mV in a whole-cell patch clamp experiment. The time dependence of the sodium
channel conductance is simplified by assuming that, when the channel activates, it has a fixed
1 5 conductance gNa = ^^Na f° r a fixed time TNa~l -0 ms > a ft er which the channel inactivates.
Using a biphasic square wave stimulus kernel (each phase has a time t\ and is repeated at a
frequency^ 1/7), the total current entering the cell during Fis:
"' dt
_ Qua ~Ql
T
(2)
20 Here, Tjsj a is the time the sodium channels are open. 7?jsj a =l/gNa * s ^ e membrane
resistance when the sodium channels are open. is the normal (leak) membrane resistance. Vl
is the leak reversal potential (i.e. the resting membrane potential). Vjq a is the sodium reversal
potential. x r is the time constant for recovery from inactivation; this is actually a function of the
hyperpolarizing voltage achieved during the pulse, but here we assume it to be a constant.
25 In reality, sodium channels from different parts of the cell experience different membrane
potential changes, and the parameters xj^a > T r » an< 3 i?Na have strong dependence upon membrane
potential. The full model would take into account the cell morphology, a random distribution of
cell orientations, and the potential and time dependence of these parameters. It would then be
possible to convolute these dependencies to produce effective values for these parameters. These
30 procedures are too involved for the present discussion. We will instead recognize that the values
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that are extracted from fits to these equations represent complicated averages of the underlying
channel properties.
Solving equation (2) for the transmembrane potential change during stimulation (V-VjJ)
yields:
( V -vJ=(v Na -v L )-f—
J 0 J
1 — exp
'- ')}
\ t rise J _
fi-
ll
Na
R L T Na
1 - exp
1 , T Naf
, where
and
(3)
If the stimulation is carried out for a long enough time such that a new transmembrane
potential is reached, the steady-state equation is:
10
15
To determine the effective release from inactivation time and open channel conductance,
experiments were conducted as described in example 8, using a biphasic square wave kernel at a
constant amplitude of 43 V/cm at varying frequencies and with pulse durations of 20 ras, 10 ms, 5
ms, 2 ms and 0.3 ms. The results, shown in FIG. 16, display the response as a function of
stimulation frequency for several pulse durations. In this case as predicted, the response saturates
at high frequencies as the transmembrane potential apparently approaches the sodium reversal
potential. To determine the effective release from inactivation time and channel open time the
response R was fitted to the modified Hill equation below.
20
Equation (5) can be derived from equation (4) by recognizing that the ratiometric response
R=l for no transmembrane potential change, and is linear in the transmembrane potential change
with an uncalibrated proportionality constant^.
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In equation (5), A and fo are adjustable parameters. The fitting was performed using a
non-linear least-squares analysis using Origin 6.0 software (Microcal, Northampton MA).
The parameters To = l/f() from equation (5) above were extracted from these fits and plotted
against the pulse duration and are shown in FIG. 17. The line in this figure is a fit to an
5 exponential decay, and from this fit, we extract the release from inactivation time constant (x r )
x r =5.7 ms and R L T Na /R Na = 0.314 .
Assuming that TN a =l ms and Rl=45 GCl, then R]Sf a =140 Mf2. This in turn means that the
peak sodium conductance would be 100mV/140MQ = 700pA . This is in excellent agreement
with the value measured in whole-cell patch clamp.
10 Example 12: Analysis of an exogenous sodium channel in a cell line with other endogenous ion
channels
Wild-type HEK-293 cells typically express a variety of endogenous potassium and
chloride currents (Zhu et al., 1998, J. Neurosci. Meth. 81:73-83), so that the resting membrane
resistance for these cells is 5-10 GCl. As a consequence the membrane time constant for these cells
15 is corresponding smaller, thus for optimal stimulation of the cells, one would predict that the
electrical stimulation protocol should be repeated at relatively higher frequencies compared to cells
without endogenous potassium channels in order to generate comparable signals.
To test that a voltage regulated sodium channel could be efficiently electrically stimulated
using the present invention in this cellular background, HEK-293 cells were stably transfected with
20 a voltage dependent sodium channel hereinafter referred to as NaV3. Cells were transfected and
selected as described in section VI and labeled with FRET dyes as described in Example 8. Cells
were plated and loaded with 15 uM CC2-DMPE and 2 uM DiSBAC6 (3) and then subjected to a
25 V/cm, biphasic stimulus train repeated at a frequency of 90 Hz and with a 5 ms/phase pulse
duration. The stimulation pulse train occurred for a total duration of 3 seconds and the digitization
25 rate for data collection was 50 Hz.
The response as a function of time (FIG. 18) shows a rapid (<20 ms rise time) initial phase
which decays with a time constant of about 40 ms to a stable plateau. A small rebound potential
change is also present between the spike and the plateau. We interpret this behavior as due to the
activation of endogenous voltage-dependent potassium channels (Ky) that occur after the first
30 stimulus pulse. Activation of these endogenous potassium channels would be expected to cause a
reduction of transmembrane potential as potassium leaves the cell consistent with the experimental
data. As electrical stimulation continues the transmembrane potential reaches a new equilibrium
which is set by the balance of sodium influx into the cell and potassium efflux out of the cell. At
the end of stimulation, the decay time constant of the response is about 143 ms, corresponding to a
35 leak resistance of about 9 GQ.
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To determine whether this overall smaller response could be reliably used for drug
discovery were conducted to determine whether the effects of TTX or tetracaine could be
accurately characterized. The results shown in FIG. 19 demonstrate that the pharmacological
inhibition profiles of these drugs using the present invention are consistent with the known
5 behavior of the NAV3 sodium channel with these agents. The dose-response curve for TTX could
be fitted with a Hill function with an EC50 = 25 nM and Hill coefficient 1.1. The dose-response
curve for tetracaine could be fitted to a curve with an EC50 =11 uM and Hill coefficient 0.97.
These results suggest that the response is caused by sodium channel activity and that
pharmacological information on known and unknown compounds can be obtained using this
10 method.
Example 13: Analysis of HEK-293 cells expressing the NaV4 sodium channel
To determine whether the present method is generally applicable to a wide range of
different sodium channels, HEK-293 cells were stably transfected with another voltage dependent
sodium channel, hereinafter referred to as NaV4. These cells were transfected, selected and loaded
15 with FRET dyes as described in section VI and Example 8. The results of a dose-response curve
for tetracaine on this channel are shown in FIG. 20. Here the data points are averages and
standard deviations of eight wells and the solid line is a fit to a Hill function with an estimated
EC50 — 35 u.M and Hill coefficient 1.35. These results are consistent with the known
pharmacology of this ion channel and demonstrate again that the cellular response is caused
20 primarily by sodium channel activity.
Example 14: Analysis of HEK-293 cells expressing a mixture of voltage-activated chloride and
potassium channels
A demonstration of the direct stimulation of voltage-dependent chloride and potassium
channels was performed using wild-type HEK-293 cells, which endogenously express a mixture of
25 several voltage-activated chloride and potassium channels (Zhu, Zhang et al. 1998). Wild-type
cells were grown in 96-well microtiter plates and assayed at confluence after staining with the
FRET dyes according to the protocol in Appendix Al. Initial stimulus parameters included a 3
second long electrical stimulation at 20 Hz with a biphasic square wave stimulus kernel with a
pulse duration of about 5 ms/phase. Stimuli were performed at varying electric field intensities to
30 determine the threshold field strength for a measurable cellular response, and in the presence or
absence of potassium channel blockers.
FIG* 21 shows the cellular voltage response obtained during this experiment. In this
figure, each panel contains the ten-second time trace of the response for a single well. The panels
are laid out to match their relative positions on the plate. The vertical axis in each panel is the
35 background subtracted, normalized fluorescence ratio of the FRET voltage sensitive dye
combination CC2-DMPE/DiSBAC2(3), changes in this quantity are roughly proportional to
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WO 02/08748 PCT/US01/21652
changes in the membrane potential. Each column had identical stimulation conditions, with
increasing electric field strength from left to right across the plate. The twelfth column of the 96
well plate (not shown) contained no cells and were used for background subtraction. Rows 6-8
contained 10 mM TEA to block the voltage dependent potassium channels. At the lowest field
5 strengths tested, there was no detectable response. At intermediate electrical fields, a negative
voltage response can be seen which rapidly decays when the stimulus is removed. At the highest
fields a large positive response is elicited. This behavior sets in above 50 V/cm, similar to the
electropermeablization threshold seen in CHO cells expressing NaVl, (Example 8).
FIG. 22 shows the response averaged between 4.5 and 5.0 seconds of stimulation as a
10 function of the electric field intensity. The large positive responses above 60 V/cm were excluded
to show the channel-dependent negative responses. The coefficient of variation of the response is
generally extremely small, yielding exceptionally large screening windows (see Appendix A3).
For the unblocked data for 20-40 V/cm, the difference between stimulated and unstimulated wells
is over 20 standard deviations.
15 Tetraethylammonium (TEA), a well-known potassium channel blocker (Hille, 1992, Ionic
Channels of Excitable Membranes), was added to rows 6, 7, and 8 at a fully-blocking concentration
of 10 mM. This treatment partially blocks the response. This result is consistent with the existence
of both potassium (blocked by TEA) and chloride (unaffected by TEA) channels in these cells that
respond to electrical stimulation. The effect of the potassium channels can be isolated by blocking
20 the chloride channels with 4,4'-diisothiocyanostilbene-2 5 2'-disulfonic acid (DIDS) or 4-acetamido-
4-isothiocyanostilbene-2,2 ? -disulfonic acid (SITS; see Hille, 1992, Ionic Channels of Excitable
Membranes). Then, the same cell line could be used to screen two channel classes.
Example 15: Identification of state dependent blockers
Any proposed screening system should preferably be able to reproduce the pharmacology
25 of known compounds as determined by accepted methods. To verify that this was the case for the
present invention, a series of test compounds of defined activity were analyzed using a CHO cell
line that expresses the NaV2 channel. To accomplish this, cells were cultured in 96 well plates and
stained with voltage sensitive dyes as described in Appendix Al. Test compounds were added to
the cells with the oxonol loading buffer. Unless otherwise noted, the compounds were tested as in
30 replicates of 8, with 1/3 dilutions across eleven columns of the assay plate.
FIG. 23 shows the time traces for selected concentrations of the sodium channel blockers
tetrodotoxin (TTX) and tetracaine.
Tetrodotoxin is a potent, reversible, non-state specific sodium channel antagonist. By
comparison tetracaine is a use dependent sodium channel blocker that exhibits different affinities
35 for different sodium channel states.
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WO 02/08748 PCT/US01/21652
The results show that the present invention provides for highly reproducible results with
relatively little variability either between samples or between plates. In FIG. 23 the effect of TTX
can be seen as a progressive loss of response, without significant changes in the shape of the
response. By comparison with tetracaine the responses not only decreases, but changes shape as the
5 concentration varies. The C.V. for these experiments were 10% (TTX) and 9% (tetracaine),
compared to typical CVs using the same voltage dyes, but traditional liquid addition were 16%
(TTX) and 18% (tetracaine).
Importantly the results also show that the present invention can identify the state
dependent blockage of the sodium channel by tetracaine. The use-dependent block of tetracaine is
10 more apparent in the dose-response curves shown in FIG. 24. For TTX, the channel block is
independent of the time window used for calculating the response. For tetracaine, however, the
blockade is an order of magnitude stronger at 3 seconds than at 1 second. Under the same
stimulation conditions, other use-dependent blockers (lidocaine and bupivicaine) showed a smaller
amount of shift in the dose-response curves. The EC50 values obtained by the electrical
15 stimulation protocol for lidocaine were similar to the high-frequency values reported in the
literature (see Table 4); this suggests that lidocaine and bupivacaine have fast enough use-
dependence to be fully saturated at the 20 Hz stimulus used here. This in turn suggests that we can
explore the use-dependent properties of local anesthetics by varying the stimulation frequency.
Table 4 lists the blocking concentrations for several sodium channel antagonists. The
20 literature values reported have all been measured using whole-cell patch clamping, and are thus
based on direct measurements of the sodium channel current.
Table 4
Pharmacology of NaV2 in the electrical stimulation assay
Compound
Electric field stimulation
Literature value
Reference
Tetracaine
0.19
Bupivacaine
1.0
Lidocaine
30
11
a
97
d
Phenytoin
24
19
a
36
d
WIN-17317
0.009
0.009
b
tetrodotoxin
0.006
0.008
c
saxitoxin
0.001
c
verapamil
3
d
capsaicin
1.6
amiloride
>1000
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WO 02/08748 PCT/US01/21652
References
a Ragsdale et al., 1996, Proceedings of the National Academy of Sciences, U.S.A. 93 : 9270-9275
b Wanner et al, 1999, Biochemistry 38: 1 1 137-1 1 146.
c West et al., 1992, Neuron 8: 59-70.
5 d Ragsdale et al., 1991, Molecular Pharmacology 40: 756-65.
In Table 4, the table entries are EC50 values (in micromolar) for fits to the dose-response
curves from each assay. Each experiment was done twice, with four wells per drug concentration
per experiment. In each experiment, eleven concentrations were used, spanning five orders of
10 magnitude in concentration. Reported values are the averages of the calculated EC50 from each
experiment. In the cases of use-dependent blockers, the lowest recorded values are quoted.
WIN- 173 17 and TTX are potent tonic blockers of a variety of sodium channels. These
compounds can be detected using the electrical stimulation format, which yields blocking
potencies near the literature values.
15 The first four drugs (lidocaine, bupivicaine, tetracaine, and phenytoin) are use-dependent
blockers. That is, they have different affinities for the various states of the channel. They are of
great therapeutic relevance, since at the proper concentration, they can block damaging repetitive
bursting of neurons and muscle cells while leaving normal, low-frequency activity unaffected. In
all cases, the measured blocking concentration measured with electric stimulation is close to the
20 reported literature value. The electrical stimulation assay format is the only reliable high-
throughput method for detecting all modulators of sodium channels, including agonists,
antagonists, and use-dependent blockers.
Example 16: Applicability for high throughput screening
For the purposes of high throughput screening, the responses should be reliable enough to
25 confidently tell the difference between active and inactive compounds. This can be quantified by
examining the distribution of the responses obtained under identical stimulation conditions,
comparing native channels with fully blocked channels. Due to experimental uncertainty and noise
in the system, there will be some scatter in the responses. We would like to be able to statistically
quantify this scatter, and use it to predict the probabilities of misidentifying responses as either
30 false positives or false negatives.
To do this a plate of cells expressing the NaV2 voltage-dependent sodium channel was
loaded with the FRET dyes. One well per column was 'randomly' spiked with 1 jliM TTX,
approximately 200 times the half-blocking concentration. The cells were assayed with a 20 Hz, 3
sec burst of 25 V/cm, 5 ms/phase, biphasic stimuli. The results are shown in FIG. 25. The wells
35 spiked with TTX can easily be distinguished by eye as the wells with little or no detectable
response.
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WO 02/08748 PCT/US01/21652
The ratiometric response two seconds after the stimulus began is shown in FIG. 26. The
two populations (blocked and unblocked) can easily be distinguished. The average blocked
response was 1.01 1 ±0.004 while the average unblocked response was 2.67 ±0.21. The coefficient
of variation for the unblocked response is 13%. The screening window (i.e. the difference between
5 the populations normalized to the standard deviations, see Appendix A3) is 7.8(crj+a2) ? where
ai=0.21 is the standard deviation of the unblocked response and (72=0.004 is the standard
deviation of the blocked response. If we take the cutoff point to distinguish blockers from
nonb lockers midway between the populations (at 1.042), then the rate of statistical false negatives
and false positives (assuming a normal distribution) is l-prob(7.75)=10~14. This suggests that
10 during a screen of a large compound library (10** compounds), the probability of encountering a
single false positive or false negative during the entire screen is only one in a million. For
comparison, if the difference between the populations were only 3 and the cutoff was optimally
placed, the false positive/negative rate would be 0.3%, a factor of 10 H higher. For an actual
screen, in which we would want to include as hits compounds which do not give complete block, a
15 tradeoff exists between detecting weak pharmacological activity and the rate of false positives. If,
for example, we desire a false positive rate of 0.1%, then in this screen we can put the screening
cutoff at 3.3 standard deviations below the mean of the unblocked response, or at 1.97. In this
case, the rate of false negatives is effectively zero, and compounds which block only 50% of the
response will be identified as hits.
20 Mathematically, there are two reasons that the blocked and unblocked populations overlap
so little. First, the coefficient of variation of the unblocked response is relatively small. That is,
each response is nearly identical to every other response. Second, and perhaps more importantly,
there is absolutely no detectable response from the blocked wells. The scatter from blocked wells
is consequently extremely small, so that we can place the boundary for distinguishing the
25 populations very low.
In assays performed using liquid addition protocols for stimulation, addition artifacts
generally give some small response with an associated scatter. The scatter of the blocked response
reduces the screening window, increases the probability of false positives and false negatives, and
limits the screener's ability to identify partial blockers.
30 Example 17: Screening in complex cell lines
The feasibility of electrical stimulation of cells expressing multiple channels was
demonstrated using cultures of the HL5 cell line. These cells were generated by immortalizing
cardiac muscle cells (Claycomb et al., 1998, PNAS 95: 2979-84). They contain several voltage-
activated sodium, calcium, and potassium channels, as well as a strong inward rectifier potassium
35 current and potassium and chloride leak currents. Cells were grown in 96-well microtiter plates
and assayed at confluence. They were stained according to the protocol in Appendix Al.
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WO 02/08748 PCT/US01/21652
Ratiometric fluorescence measurements were made during electrical stimulation using VIPR™ as
described above, and the data were analyzed according to the procedures in Appendix A2.
Stimulus parameters were arbitrarily chosen to be: 3 second long burst at 10 Hz with a biphasic
square wave stimulus kernel with a pulse duration of 5 ms/phase. Stimuli were performed at
5 varying electric fields to determine the threshold field. Two rows of wells contained 10 |uM TTX
to partially block the cardiac sodium channel, and two rows contained 10 raM TEA to block the
voltage-dependent potassium channels. FIG. 27 shows the normalized responses of each well.
Generally as the electric field strength increases, the cellular response increases. The last three
columns show signs of electropermeablization as the voltage continues to increase. In columns
10 6,7, and 8, the ratio actually rebounds below the starting ratio, suggesting an after-
hyperpolarization (a phenomenon caused by slow closing of voltage-dependent potassium
channels).
The rate of the cellular response is extremely fast, and may be apparently limited by the
ability of the ethyl oxonol to rapidly redistribute within the membrane. The rapid response is
1 5 consistent with a high resting conductance of the cell due to the leak currents and the expression of
potassium inward rectifier channels. TTX partially blocks the positive response, indicating that it
is at least partially due to the voltage-dependent sodium current.
FIG. 28 shows the response of the untreated cells (rows 1 -4) as a function of the applied
electric field. The response increases sigmoidally with the electric field. Above 50 V/cm, there is
20 a sustained signal which is unaffected by TTX. As discussed previously, this behavior is
consistent with the electropermeablization of the cellular membrane at high electric field strengths.
Also shown in FIG. 28 is the screening window (see Appendix A3) as a function of the stimulus
field.
These results demonstrate that HL5 cells can be effectively assayed using the electrical
25 stimulation technique. Compounds which are known to modify different ion channels cause
detectable changes in the response. Because these ion channels are identical to those expressed by
the heart, such an assay would be useful as a secondary screen, to eliminate or mark for
modification those compounds which may interfere with normal heart function. It could also be
useful as a primary screen, to discover compounds which may have desirable effects on any one
30 (or a combination) of the heart ion channels.
Example 18: Electrical stimulation of cell cultures using surface electrodes.
Surface mounted electrodes were prepared on glass coverslips coated with chromium (as
an adhesion layer) and gold (as a conductive layer). The metallized coverslips were custom-built
by Thin Film Devices, Inc. (Anaheim CA). The coverslips were one inch square, 0.17 mm thick
35 Corning 7059 glass. Metallization was performed by vacuum sputtering deposition. The
chromium layer was approximately 1000 A thick, and served as an adhesion layer. The gold layer
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WO 02/08748 PCT/US01/21652
was approximately 5000 A thick, and served as a conductive layer. The resistivity of the deposited
metal was less than 0.1 Q/square. A 4-mm gap was etched through the metal by hand-masking the
metal surface with a chemically-resistant polymer (SI 400-27, Shipley Co., Marlborough MA),
then etching through the metal layers with five minutes in Gold Etchant TFA, followed by five
5 minutes in Chromium Etchant TFD (Transene Co., Danvers MA). The coverslips were attached to
the bottoms of 96 well plates with silicone elastomer (Sylgard 184 (Corning), cured 90 minutes at
70 °C). After sterilizing with 365 nm UV irradiation for 30 minutes and coating with the cell
adhesion molecule poly-D-lysine (molecular weight 300,000, 1 mg/mL in Dulbecco's phosphate
buffered saline for 30 minutes, then rinsed 3 times with distilled water), living cells could be
10 successfully grown and cultured on the electrode surfaces.
To validate the surface electrode stimulator CHO cells at an initial density of
approximately 1000 cells/mm^ were plated into the wells of the 96 well plate and left to attach for
approximately 16 hours. These cells were transfected to express a potassium channel, which set
the transmembrane potential to around -80 mV, and the NaV3 sodium channel. After reaching
15 confluence, the cells were loaded with the voltage-sensitive FRET dye combination of CC2-DMPE
and DiSBAC2 (3) as described in Appendix Al. The metal surface electrodes were connected to
the output of a pulse generator, which in this case was an exponential-decay electroporator (Gene
Pulser II, Bio-Rad Corp., . Hercules CA). Ratiometric fluorescence imaging was performed on a
Zeiss Axiovert TV microscope, equipped with a 75 W xenon arc lamp light source. Excitation
20 light was filtered using a 405±10 nm dielectric interference filter and a 445 DXCR dichroic mirror.
Emission light was split with a second 525XR dichroic mirror, and measured with a pair of
Hamamatsu HC124 photomultiplier tubes (PMTs). One PMT had a 475±40 nm dielectric
interference filter in front of it to monitor the blue fluorescent signal. The second PMT had a
580±35 nm dielectric interference filter in front of it to monitor the orange fluorescent signal. The
25 optical filters and dichroic mirrors were purchased from Chroma Technology Corp., Battleboro
VT. Ratiometric fluorescence imaging was performed on fields containing approximately 100
cells. Correction for background fluorescence was performed by measuring the blue and orange
signals in a field with no cells, then subtracting these from the signals obtained from the cells.
Then the ratiometric signal, proportional to the transmembrane potential changes, was calculated
30 as described in Appendix A2.
The stimulation protocol used single, monophasic electric field pulses of variable amplitude.
The pulses were exponential-decay waveforms with a 4.3 ms decay time constant. The amplitude at
the beginning of the pulse was varied from zero to 56 V/cm.
A typical voltage response for CHO cells expressing a potassium channel and the NaV3
35 sodium channel after a three separate 45 V/cm stimulation responses are shown in FIG. 29 for the
same field of cells, demonstrating repeatability of the response. The speed of the response in this case
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WO 02/08748 PCT/US01/21652
is limited primarily by the response time of the mobile hydrophobic dye, which for the ethyl oxonol
used is about 0.5 second.
The average ratiometric response of a population of cells grown in a 96 well multiwell plate
stimulated with monophasic stimuli of varying field strengths is shown in FIG. 30. The points in this
5 curve are the average peak response of 4 stimulations on the same culture. As is to be expected from
an action-potential-type response curve, there is no detectable response below about 18 V/cm. The
threshold region is relatively narrow. Between about 20 and 40 V/cm the response increases with
increasing field strength. Above 40 V/cm the response plateaus.
Example 19: Analysis of wild-type RBL cells expressing IRK1
10 Rat basophilic leukemia (RBL) cells endogenously express the potassium inward rectifier
channel IRK1 (Wischmeyer et al, Pflugers Arch. 429:809-819, 1995). This channel selectively
conducts potassium ions, with a highly non-linear conductance characteristic. The conductance is
nearly linear below the potassium reversal potential Vyj and rapidly drops to near zero beginning
at about 10 mV positive of Vj^. Cells expressing large amounts of inward rectifier channels tend to
15 have resting transmembrane potentials within a few millivolts of
On the side of the cell where the transmembrane potential is driven positive by an external
electric field applied to cells expressing IRK1 and few other ion channels, the IRK1 channels will
rapidly close and cease conducting. On the side of the cell where the transmembrane potential is
driven negative, the IRK1 channels will open and pass potassium current. If this side of the cell is
20 driven sufficiently negative, so that the local transmembrane potential is more negative than Vj^, a
net inward potassium current will exist. This current will cause a positive global transmembrane
potential change. Because the IRK1 channel does not inactivate, this current should be sustained
for as long as the external field is applied.
Adherent RBL cells were seeded into 96-well plates and loaded with FRET dyes as
25 described in Appendix Al. Three rows of wells contained 400 \iM barium chloride to block the
IRK1 channel. The plates were analyzed using a VIPR™ reader while being electrically
stimulated with a biphasic stimulus train repeated at a frequency of 50 Hz and with a 5 ms/phase
pulse duration. The stimulation pulse train occurred for a total duration of 5 seconds and the
digitization rate for data collection was 50 Hz. The applied electric field was fixed for each
30 column of eight wells, and was varied from 7.2 to 72 V/cm. The data were analyzed according to
the procedures in Appendix A2. The normalized ratio after three seconds of stimulation was
calculated, averaged for the two population of wells (with and without barium block), and plotted
as a function of the applied field in FIG, 31. The error bars are standard deviations of the
responses. Open squares are the responses without barium block; solid circles are the responses
35 with barium block. The data from the wells with barium block indicate that there is no detectable
voltage change during stimulation until the field reaches 80 V/cm, at which point some
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WO 02/08748 PCT/US01/21652
electropermeablization may be occurring. The unblocked wells show nearly linear response above
a threshold at around 20 Y/cm. This example clearly shows that the present invention can be used
to modulate the transmembrane potential in either positive or negative directions, depending upon
the stimulus parameters and the properties of the ion channels expressed by the cell.
5 The present invention expands the applicability of electrical stimulation to include non-
excitable cells, by providing instrumentation and methods that enable effective stepwise control of
membrane potential without resulting in significant electroporation. The present invention
achieves this result via the use of highly uniform, repetitive pulses of electrical stimulation applied
to the medium surrounding the cells. The applied electric fields typically do not directly alter the
10 average transmembrane potential of the cell, but instead create symmetric positive and negative
transmembrane potential changes on the sides of the cell facing the cathode and the anode,
respectively.
The approach exploits the ion selectivity and the non-linear gating and conductance
characteristics of voltage-dependent ion channels. The approach also exploits the fact that typical
1 5 intact cells have long time constants for decay of transmembrane potential changes. Even in those
cases where the charge injected into the cell by a single stimulus pulse is too small to be detected
reliably, appropriately applied multiple stimulus pulses can build large net transmembrane
potential excursions. By varying the number, duration, and the shape and amplitude of the pulses,
it is possible to artificially set, or change the transmembrane potential of living cells in a fashion
20 that is similar to patch clamping. Other channels, leak currents or transporters that are not
classically considered voltage-dependent, can also be assayed by inducing transmembrane
potential changes using a second, voltage-dependent channel and detecting the current flow or
transmembrane potential changes as a result of activation of the target channel or transporter.
The present method is robust, compatible with optical detection methodologies and readily
25 amendable to a wide range of potential applications including high throughput screening for use in
drug discovery. In many assay formats direct electrical stimulation avoids the requirement for liquid
addition, making the assay simpler. Complex manipulations of the transmembrane potential can
readily be accomplished using variations in the stimulation protocol. Thus, virtually any voltage-
sensitive channel can be induced to open regardless of the state of inactivation or voltage
30 dependency. For high throughput drug discovery this relaxes the requirements for specialized cell
types, and allows assays to be rapidly performed with readily available cell lines.
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APPENDICES
PCT/US01/21652
Al . Staining protocol of Voltage FRET dyes
Reagents:
5
Assay buffer #1:
140 mMNaCl
4.5 mM KC1
2 mM CaCl2
10 lmMMgCl 2
10 mM HEPES
10 mM glucose pH 7.40, 330 mOs/kg
Pluronic stock (1000X):
15 100 mg/mL pluronic 1 27 in dry DMSO
Oxonol stock (3333X):
10 mM DiSBAC 2 (3) in dry DMSO
20 Coumarin stock (1 000X):
10 mM CC2-DMPE in dry DMSO
ESS-CY4 stock (400X):
200 mM ESS-CY4 in water
25
Loading and Assay Protocol
1. Preparation of CC2-DMPE loading buffer. Normally for a 96-well plate, 10 mL of staining
solution will be prepared per plate,
i) Mix equal volumes (10 |iL) of coumarin stock and pluronic stock in a tube.
30 ii) Add 10 mL Assay Buffer #1 to tube while gently vortexing.
Loading concentration: 10 juM CC2-DMPE and 0.1 jag/ml pluronic.
2. Prepare oxonol loading buffer:
i) Mix equal volumes (3.3 juL) of oxonol stock and pluronic stock in a tube.
ii) Add 10 mL Assay Buffer #1 to tube while gently vortexing.
35 iii) Add 25 uL ESS-CY4 while vortexing.
Loading concentration: 3 jjM DiSBAC2(3), 0.2 jug/ml pluronic, and 0.5 mM ESS-CY4.
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WO 02/08748 PCT/US01/21652
iv) If required, combine test compounds with the loading buffer at this time.
3. Rinse cells twice with Assay Buffer #1, removing all fluid from wells each time.
4. Add 100 juL CC2-DMPE loading buffer to each well. Incubate 30 minutes at room temperature,
avoiding bright light.
5 5. Rinse cells twice with Assay Buffer #1, removing all fluid from wells each time.
6. Add 100 |LiL oxonol loading buffer to each well.
7. Incubate for 30 minutes at room temperature avoiding bright light. Use immediately.
10 A2. Analysis of VIPR™ reader data
Data were analyzed and reported as normalized ratios of intensities measured in the 460 nm and
580 nm channels. The process of calculating these ratios was performed as follows. On all plates,
column 12 contained Assay Buffer #1 with the same DiSBAC2(3) and ESS-CY4 concentrations as
used in the cell plates, however no cells were included in column 12. Intensity values at each
1 5 wavelength were averaged in initial (before the stimulus) and final (during the stimulus) windows.
These average values were subtracted from intensity values averaged over the same time periods in
all assay wells. The ratios obtained from samples in the initial (Ri) and final windows (Rf) are
defined as:
20 (intensity 460 nm, initial - background 460 nm , initial)
Ri= (A2 .i)
(intensity 580 nm, initial - background 580 nm, initial )
25 (intensity 460 nm, final - background 460 nm, final)
Rf= ( A2 .2)
(intensity 580 nm, final - background 580 nm, final)
Final data are normalized to the starting ratio of each well and reported as Rf/Ri.
30
A3. Screening Window
The screening window W for a response is defined as follows. Data from multiple wells at
identical stimulus conditions are required. The control wells can either be pharmacologically
35 blocked or untransfected cell stimulated with the full electric field. Alternatively, one might use
transfected cells with no stimulus applied.
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Responses from experimental and control wells are measured. The average and standard
deviations of the responses in the experimental (R±AR) and control (C±AC) wells are calculated.
The screening window is defined as the difference between experimental and control signals
normalized to the sum of the standard deviations.
R-C
A# + AC
(A3.1)
10
A general rule of thumb for an acceptable screening window is W>3. This allows one to choose a
cutoff line midway between control and experimental responses which ensures a false
negative/positive rate less than 1%. Assuming a normal distribution, the false positive/negative
rate as a function of the screening window W is:
15 Table A3.1. The faise positive/negative rate P(W) as a function of the screening window W as
defined in Equation A3.1. This calculation assumes that the cutoff for identification of a hit is
placed an equal number of standard deviations away from the positive and negative control
responses.
w
P(W)
1
0.3173
2
0.0455
3
0.0027
4
6.334E-5
5
5.733E-7
6
1.973E-9
7
2.559E-12
8
1.221E-15
9
<1E-18
10
<1E-18
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WO 02/08748 PCT/US01/21652
WHAT IS CLAIMED IS :
1. A method of characterizing the biological activity of a candidate compound
comprising:
exposing one or more cells to said compound;
5 repetitively exposing said one or more cells to one or more electric fields so as to
effect a controlled change in transmembrane potential of said one or more cells; and
monitoring, without using a patch clamp, changes in the transmembrane potential
of said one or more cells.
2. The method of Claim 1, wherein said monitoring comprises detecting fluorescence
10 emission from an area of observation containing said one or more cells.
3. The method of Claim l 9 wherein said electric fields are biphasic.
4. The method of Claim 3, additionally comprising limiting spatial variation in
electric field intensity so as to minimize irreversible cell electroporation.
5. The method of Claim 1, wherein one or more electrical fields cause an ion channel
15 of interest to cycle between different voltage dependent states.
6. The method of Claim 5, wherein said one or more electrical fields cause an ion
channel of interest to open.
7. The method of Claim 5, wherein said one or more electrical fields cause an ion
channel of interest to be released from inactivation.
20 8. The method of Claim 1, wherein said one or more cells comprise a voltage sensor
selected from the group consisting of a FRET based voltage sensor, an electrochromic
transmembrane potential dye, a transmembrane potential redistribution dye, an ion sensitive
fluorescent or luminescent molecule and a radioactive ion.
9. The method of Claim 1, wherein said one or more cells comprise a voltage
25 regulated ion channel.
10. The method of Claim 9, wherein said voltage regulated ion channel is selected
from the group consisting of a potassium channel, a calcium channel, a chloride channel and a
sodium channel.
11. The method of Claim 1, wherein said electric field exhibits limited spatial
30 variation in intensity in the area of observation of less than about 25% from a mean intensity in
that area.
12. The method of Claim 1 1, wherein said one or more electrical fields varies over an
area of observation by no more than about 15 % from the mean electrical field at any one time.
13. The method of Claim 12, wherein said one or more electrical fields varies over an
35 area of observation by no more than about 5 % from the mean electrical field at any one time.
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WO 02/08748 PCT/US01/21652
14. The method of Claim 1, wherein said one or more electrical fields comprises
stimulation with either a square wave-form, a sinusoidal wave-form or a saw tooth wave-form.
15. The method of Claim 1, wherein said one or more electrical fields have an
amplitude within the range of about 10 V/cm to about 100 V/cm.
5 16. The method of Claim 15, wherein said one or more electrical fields have an
amplitude within the range of about 20 V/cm to about 80 V/cm.
17. The method of Claim 1, wherein said one or more electrical fields are repeated at a
frequency of stimulation that is greater than or equal to the reciprocal of the transmembrane time
constant of said one or more cells.
10 18. The method of Claim 1, wherein said one or more electrical fields are repeated at a
frequency of stimulation within the range of zero to 1kHz.
19. The method of Claim 1, wherein said one or more electrical fields have a pulse
duration within the range of about 100 microseconds to about 20 milliseconds.
20. The method of Claim 1, wherein said transmembrane potential is developed across
15 the plasma membrane of said one or more cells.
21. A method of assaying the biochemical activity of a compound against a target ion
channel comprising:
selecting a cell line having a normal resting transmembrane potential
corresponding to a selected voltage dependent state of said target ion channel;
20 expressing said target ion channel in a population of cells of said selected cell line;
exposing said population of cells to said compound;
repetitively exposing said population of cells to one or more electric fields so as to
effect a controlled change in transmembrane potential of said one or more cells; and
monitoring changes in the transmembrane potential of said one or more cells.
25 22. The method of Claim 21, wherein said target ion channel is exogenously expressed
in said cell line.
23. The method of Claim 21, wherein said cell line is transfected with nucleic acid
encoding said target ion channel.
24. The method of Claim 23, wherein said cell line expresses insignificant levels of
30 other ion channels.
25. The method of Claim 24, wherein said cell line is selected from the group
consisting of CHL, LTK(-), and CHO-K1.
26. The method of Claim 21 wherein said target ion channel is a sodium channel, and
wherein said population of cells is selected from the group consisting of CHL cells, LTK(-) cells,
35 and CHO-K1 cells.
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WO 02/08748 PCT/US01/21652
27. The method of Claim 21 wherein said target ion channel is a sodium channel, and
wherein said population of cells is selected from the group consisting of HEK-293 cells, RBL cells,
Fl 1 cells, and HL5 cells.
28. The method of Claim 21 wherein said target ion channel is a potassium channel,
5 and wherein said population of cells is selected from the group consisting of CHL cells, LTK(-)
cells, and CHO-K1 cells.
29. The method of Claim 21 wherein said target ion channel is a calcium channel, and
wherein said population of cells is selected from the group consisting of CHL cells, LTK(-) cells,
and CHO-K1 cells.
10 30. A method of assaying ion channel activity comprising:
exposing at least one cell to a plurality of electric field pulses so as to create a
controlled change in transmembrane potential and so as to activate an ion channel of
interest; and
detecting ion channel activity by detecting one or more changes in transmembrane
1 5 potential without using a patch clamp.
31. The method of claim 30, wherein said at least one cell comprises a voltage sensor
selected from the group consisting of a FRET based voltage sensor, an electrochromic
transmembrane potential dye, a transmembrane potential redistribution dye, an ion sensitive
fluorescent or luminescent molecule and a radioactive ion.
20 32. The method of Claim 31 wherein said voltage sensor comprises a FRET based
voltage sensor.
33. The method of Claim 32, wherein said ion channel of interest is a voltage
regulated ion channel.
34. The method of Claim 33, wherein said plurality of electric field pulses cause said
25 ion channel of interest to cycle between different voltage dependent states.
35. The method of Claim 30, wherein said at least one cell is an eukaryotic cell.
36. The method of Claim 30, wherein said at least one cell is a non-excitable cell.
37. The method of Claim 30, wherein said at least one cell is a prokaryotic cell.
38. The method of Claim 30, wherein said at least one cell is a tissue culture cell.
30 39. The method of Claim 30, wherein said at least one cell is a primary cell line.
40. The method of Claim 30, wherein said at least one cell is part of an intact living
organism.
41. A method of assaying ion channel activity comprising:
expressing a selected target ion channel in at least one cell;
35 expressing a selected counter ion channel in said at least one cell;
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WO 02/08748 PCT/US01/21652
exposing said at least one cell to a plurality of electric field pulses so as to create a
controlled change in transmembrane potential and so as to activate said counter ion
channel; and
monitoring the transmembrane potential of said at least one cell.
5 42. The method of Claim 41 , wherein a transmembrane potential change is detected
when said ion channel of interest is blocked.
43. The method of Claim 42, wherein said ion channel of interest comprises a ligand
gated ion channel.
44. The method of Claim 43, wherein said counter channel comprises a sodium
10 channel.
45. A method of modifying the transmembrane potential of a cell comprising
repetitively applying biphasic electric field pulses to said cell, wherein said pulses have a
maximum amplitude of less than approximately 90 V/cm, wherein said pulses are applied at a rate
of at least about 1 per second, and wherein the total duration of each pulse is at least about 1
15 millisecond.
46. The method of Claim 45, wherein said maximum amplitude is approximately 20 to
40 V/cm.
47. The method of Claim 45, wherein said pulse duration is approximately 2 to 10
milliseconds per phase.
20 48. The method of Claim 45, wherein said pulses are applied at a rate of
approximately 20 to 100 pulses per second.
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WO 02/08748
PCT/USO 1/2 1652
FIG. 1A
immiitiiii
I I I I 11 I I 1 I I I I I I I I I I i WJJ
FIG. IB
1/35
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PCT/USO 1/2 1652
2/35
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PCT/USO 1/2 1652
200
240
-i 1
)( )( )( )(
)(
)< pi
( )( )( )( )(
T
\ /V / \ / V / V
TIP!
( )( )( )( )(
x
HI
i )i n h r
Z
HI
}(
"DEI
nnrrr
7T
HI]
< x x )( X
in
I \( K M k
x
7HFI
ni
( \{ K )( )(
i n n >( k
T
FIG. 2 A
3/35
WO 02/08748
PCT/USO 1/2 1652
200
f 1
<UUL
_
X
JL
m
T
T
7T
X
X
X
\ )\ )\ )\ l\ t\ >\ >\ /
DEI
X
X
X
< X >< X K )(
nnr
T
T
x
__
I
X
X
nnr
T
X
X
■
T
){
< )( )(
)(
)(
TTT.
210
230
/
240
FIG. 2B
4/35
WO 02/08748
PCT/USO 1/2 1652
200
220
240
210
/
«t \{ \i )l )(
X
)(
)( )
)(
( )( )( )( )(
)(
)( )
( )( )( )( )(
){
A
A
( \l \l \( \i
i n \( n k
T
-M
( n n )( )(
X
X
( )( )( )( )(
x
x
in
i n n )( n
x
T7! l'( l( J(
■QHH1
205
230
FIG. 2C
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PCT/USO 1/2 1652
FIG. 2D
6/35
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FIG. 3
7/35
PCT/USO 1/2 1652
— 1 — I —
T 1 1 1 1 1 1 1 1 1 ' 1 1 1 1 1 '
-rrrrrWr'H
— ■ — r
ii
"i — i — i — i — « — i — i — i — 1 — i — 1 — i — ■ — I — 1 — I r ~Z
jUjLLjLiLi^jLj
"1 > 1 ' 1 —
50 100 150
time (ms)
FIG. 4
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WO 02/08748
PCT/US01/21652
420
E2
E1
430
400
410
440
460
E4
470
TIME
E3
450
FIG. 5
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PCT/USO 1/2 1652
10/35
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735 745
FIG. 7
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FIG. 8
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FIG. 9
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PCT/US01/21652
o
■ mmmm
8
8
O
i
N
1.6-
1.5-
1.4-
1.3-
1.2
1.1.
1.0-
0.9.
-i — r
□
10V/cm
o
13V/cm
A
16V/cm
V
25 V/cm
O
29 V/cm
<
32 V/cm
1 — 1 — I — I — I — I — I — I — I — I — I — I — I — • — I — 1 — 1 —
0 2 4 6 8 10 12 14 16
time (s)
FIG. 10
14/35
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PCT/US01/21652
i 1 1 1 1 • 1 1 r
1 1 i 1 1 1 1 • 1 1 1
0 20 40 60 80 100
E (V/cm)
FIG. 11
15/35
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PCT/USO 1/2 1652
(f)
2.0
1.8-
1.6-
Q 1.4A
Q.
1.2-
1.0-
q i i mrw| i i i mh i | i i rrrm| i i rmrrq i i rrnTq i
n c
3 — I
■a — h -
iij i i i 1 1 n i | i iimii| i i i mm i ii i i i uj i i i mill i
0.01 0.1 1 10 100 1000
TTX(nM)
FIG. 12
16/35
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PCT/US01/21652
CD
CO
a
1.6-
1.4-
1.2-
1.0-
stim duration
□ 20 ms
i ■ ■ ■ i
I I I I I l l | I I I I
iu i r-TTTTrq i t i i 1 1 ii |
H i ill
1
10
100
1000
stimulus frequency (Hz)
FIG. 13
17/35
WO 02/08748
PCT/USO 1/2 1652
g
8
8
1
o
3
N
O
□
OV/cm
o
43V/cm
A
A
85 V/cm
V
140V/cm
I H I I I I
1 1 1 1 1 1 1 1 ii rrttl I TT I _
4 6
time (s)
T"
8
— 1—
10
FIG. 14
18/35
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0 40 80 120 160
E (V/cm)
FIG. 15
19/35
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■ nil i i rmwi i i i i mi) i i rrTrrq i i ii in i |
0.1 1 10 100 1000
burst frequency (Hz)
FIG. 16
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1E-4 1E-3 0.01
pulse duration (s)
FIG. 17
21/35
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2
8
C
8
i
O
q=
-o
CD
N
Q 0.98
time (sec)
FIG. 18
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PCT/US01/21652
1.15.
(f)
1.10-
8.
% 1-05H
1.00-
mn| i t i 1 1 ui| i i i i i i i i } < ii i rrtq i i i nni|
M i ll I III I I 1 1 1""
0.1 1
i i i i i mm i i i i inn
10 100 1000
TTX(nM)
m i i i mm i i i iii i i | i m i i i rrtvi] i i i rrttq
0.01 0.1 1 10 100 1000
tetracaine (jiM)
FIG. 19
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PCT/USO 1/2 1652
FIG. 20
24/35
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\
PCT/USO 1/2 1652
FIG. 21
25/35
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1.0-
<Z>
C
8. 0.9
0.8.
0
□
□ no blockers
• with TEA
20 40
E ( V/cm)
60
FIG. 22
26/35
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PCT/US01/21652
O
2
i
N
2.5
2.0-
1.5-
i8:
2.0-
1.5
1.0-1
-i — r
-t — \ — i — r
i i i
10 nMTTx
— i — ■ — i — ■ — i — 1 — r-
0 2 4 6 8
time (s)
i i i i i i i ■
100 nMTTx
10
T
A
O
8
8
O
"8
N
8
2.5-
2.0-
1.5-
1.0-
2.5
2.0-
1.5-
1.0-
-i — i — ' — i_j — r
T « 1 » 1 « 1 *-
1 nMtet
2
T ■ 1 « 1 1 1 >"
I ' 1 ■ 1 ' 1 r "
4 ' 6 ' 8 10
time (s)
lOOpMtet
t— « — r
B
FIG. 23
27/35
WO 02/08748
PCT/USO 1/2 1652
1.2
1.0-1
0.8
CD
I 0M
SB 0.4
0.2-
0.0-
0.01 0.1
time after stim. begins:
• 1 sec 10^=5.6 nM
A 2 sec 10^=7.9 nM
v 3 sec 10^=8.8 nM
1 10 100 1000
TTX(nM)
A
1.2
CD
CO
0.0-
1 r- , ,
time after stim. begins:
■ 1 sec 10^=1.9^
o 2 sec 10^=500 nM
A 3 sec 10^=290 nM
0.01 0.1 1
tetracaine (faM)
B
FIG. 24
28/35
WO 02/08748
PCT/USOl/21652
r '1
A
J V
r\
r\
J N
J vj
r\
j N
r\
j \
r\
J v
/ \
K
/ x
J N
A
J V
J v
r\
r\
^A
A
-J
A
An
A
A
A
rv
A
A
A
A
A
:A
A
A
A
As
:
:A
AJ
a
A
A
rv
A
A
Pv
:A
a
A
A
rv
A
:A
A
a
A
A
A
A
A
Normalized
fluorescence
ratio
time
FIG. 25
29/35
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3.0-
2.5-1
CD
S 2.0-1
o
CL
1 1
5-
1.0-
o o o o o o o oooo-
80
0
20
1 |
40 60
well number
FIG. 26
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WO 02/08748
PCT/USO 1/2 1652
10 sec
increasing field strength —
Normalized
fluorescence
ratio
time
FIG. 27
31/35
WO 02/08748 PCT/US01/21652
^-i 1 1 1 1 1 1 1 « 1 « 1 U
0 20 40 60 80 100 120
E ( V/cm)
FIG. 28
32/35
FIG. 29
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0 20 40 60
E (V/cm)
FIG. 30
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CD 1.4 J
CO
E(V/cm)
FIG. 31
35/35
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